&#34;Polyunsaturated Fatty Acid-Containing Oil Product and Uses and Production Thereof&#34;

ABSTRACT

The present invention includes a solid fat composition that includes an oil having saturated fat and a microbial oil having a long chain polyunsaturated fatty acid and an emulsifier. In particular, the solid fat composition can have high levels of long chain polyunsaturated fatty acid and low amounts of emulsifiers. In preferred embodiments, the polyunsaturated oil is an unwinterized microbial oil. The invention also relates to methods for making such compositions and food, nutritional, and pharmaceutical products comprising said compositions. The present invention also includes a microbial oil product prepared by extracting an oil-containing fraction comprising at least one LC-PUFA from a microbial biomass, and treating the fraction by a process of vacuum evaporation, wherein the oil product has not been subject to one or more of a solvent winterization step, a caustic refining process, a chill filtration process, or a bleaching process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/428,277, filed Jun. 30, 2006, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/695,996 filedJul. 1, 2005, and to U.S. Provisional Patent Application Ser. No.60/738,304, filed Nov. 18, 2005. Each of the foregoing applications isincorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

The invention relates to a polyunsaturated fatty acid-containing oilproduct and uses thereof, such as in a solid fat composition thatincludes a microbially-derived long chain polyunsaturated fatty acid anda thickener. The invention also relates to methods for making suchproducts and food, nutritional, and pharmaceutical products comprisingsaid compositions.

BACKGROUND OF THE INVENTION

It is desirable to increase the dietary intake of many beneficialnutrients. Particularly beneficial nutrients include fatty acids such asomega-3 and omega-6 long chain polyunsaturated fatty acids (LC-PUFA).Omega-3 PUFAs are recognized as important dietary compounds forpreventing arteriosclerosis and coronary heart disease, for alleviatinginflammatory conditions and for retarding the growth of tumor cells.Omega-6 PUFAs serve not only as structural lipids in the human body, butalso as precursors for a number of factors in inflammation such asprostaglandins, and leukotrienes. An important class of both the omega-3and the omega-6 PUFAs is long chain omega-3 and the omega-6 PUFAs.

Fatty acids are classified based on the length and saturationcharacteristics of the carbon chain. Short chain fatty acids have 2 toabout 6 carbons and are typically saturated. Medium chain fatty acidshave from about 6 to about 14 carbons and are also typically saturated.Long chain fatty acids have from 16 to 24 or more carbons and may besaturated or unsaturated. In longer chain fatty acids there may be oneor more points of unsaturation, giving rise to the terms“monounsaturated” and “polyunsaturated,” respectively. Long chain PUFAs(LC-PUFAs) having 20 or more carbons are of particular interest in thepresent invention.

LC-PUFAs are categorized according to the number and position of doublebonds in the fatty acids according to a well understood nomenclature.There are two main series or families of LC-PUFAs, depending on theposition of the double bond closest to the methyl end of the fatty acid:the omega-3 series contains a double bond at the third carbon, while theomega-6 series has no double bond until the sixth carbon. Thus,docosahexaenoic acid (“DHA”) has a chain length of 22 carbons with 6double bonds beginning with the third carbon from the methyl end and isdesignated “22:6 n-3”. Other important omega-3 LC-PUFAs includeeicosapentaenoic acid (“EPA”) which is designated “20:5 n-3,” andomega-3 docosapentaenoic acid (“DPA”) which is designated “22:5 n-3.”Important omega-6 LC-PUFAs include arachidonic acid (“ARA”) which isdesignated “20:4 n-6,” and omega-6 docosapentaenoic acid (“DPA”) whichis designated “22:5 n-6.”

De novo or “new” synthesis of the omega-3 and omega-6 essential fattyacids does not occur in the human; however, the body can convert theseessential fatty acids, when obtained in the diet, to LC-PUFAs such asDHA and ARA although at very low efficiency. Both omega-3 and omega-6fatty acids must be part of the nutritional intake since the human bodycannot insert double bonds closer to the omega end than the seventhcarbon atom counting from that end of the molecule. Thus, all metabolicconversions occur without altering the omega end of the molecule thatcontains the omega-3 and omega-6 double bonds. Consequently, omega-3 andomega-6 acids are two separate families of fatty acids since they arenot interconvertible in the human body.

Over the past twenty years, health experts have recommended diets lowerin saturated fats and higher in polyunsaturated fats. While this advicehas been followed by a number of consumers, the incidence of heartdisease, cancer, diabetes and many other debilitating diseases hascontinued to increase steadily. Scientists agree that the type andsource of polyunsaturated fats is as critical as the total quantity offats. The most common polyunsaturated fats are derived from vegetablematter and are lacking in long chain fatty acids (most particularlyomega-3 LC-PUFAs). In addition, the hydrogenation of polyunsaturatedfats to create synthetic fats has contributed to the rise of certainhealth disorders and exacerbated the deficiency in some essential fattyacids. Indeed, many medical conditions have been identified asbenefiting from omega-3 supplementation. These include acne, allergies,Alzheimer's, arthritis, atherosclerosis, breast cysts, cancer, cysticfibrosis, diabetes, eczema, hypertension, hyperactivity, intestinaldisorders, kidney dysfunction, leukemia, and multiple sclerosis. Ofnote, the World Health Organization has recommended that infant formulasbe enriched with omega-3 fatty acids.

The conventionally used polyunsaturates are those derived from vegetableoils, which contain significant amounts of omega-6 (C18:2 n-6) butlittle or no omega-3. While omega-6 and omega-3 fatty acids are bothnecessary for good health, it is recommended that they be consumed in abalance of about 4:1. Principal sources of omega-3 are flaxseed oil andfish oils. The past decade has seen rapid growth in the production offlaxseed and fish oils. Both types of oil are considered good dietarysources of omega-3 polyunsaturated fats. Flaxseed oil contains no EPA,DHA, DPA or ARA but rather contains linolenic acid (C18:3 n-3), abuilding block enabling the body to manufacture EPA. There is evidencehowever that the rate of metabolic conversion can be slow and unsteady,particularly among those with impaired health. Fish oils varyconsiderably in the type and level of fatty acid composition dependingon the particular species and their diets. For example, fish raised byaquaculture tend to have a lower level of omega-3 fatty acids than thosein the wild. Furthermore, fish oils carry the risk of containingenvironmental contaminants commonly found in fish. In light of thehealth benefits of such omega-3 and omega-6 LC-PUFAs (chain lengthgreater than 20), it would be desirable to supplement foods with suchfatty acids.

Liquid oils such as fish oils and certain microbial oils are known tocontain a high content of LC-PUFAs. However, due to theirpolyunsaturated nature, these oils are not solid at room temperature(i.e., 20° C.), rather being in an oil, or liquid, form. However, solidforms of PUFA-rich oils are desirable for use in certain foodapplications where liquid oils are not applicable. To form a solidcomposition, a number of approaches have been tried. A common processused to solidify unsaturated oils consists of partial or fullhydrogenation of such oils, so as to obtain semi-solid oils. Yet, as aresult of this chemical transformation, the oils become saturated andlose their healthy properties. The partial hydrogenation process alsoresults in the formation of “trans”-fatty acids, which have been shownto possess several adverse properties. Hence, by solidifying unsaturatedoils using a hydrogenation process, the beneficial properties of theunsaturated oils are substituted by the highly undesirable adverseproperties of the saturated oils and the formation of “trans”-fattyacids. Other methods include mixing the unsaturated oils with “hard” orsaturated fats so that the mixture is a semi-solid oil. Again, thebenefits of the “healthy” unsaturated oil are at least partially offsetby the presence of hardened, or saturated, fats. Other methods forforming a spreadable, semi-solid fat composition comprising high levelsof polyunsaturated fats include using high levels of particular types ofemulsifiers, or other thickeners such as fatty alcohols. Until thepresent invention, there was lacking in the art compositions comprisinga solid or semi-solid fat or food product containing high levels ofPUFAs, but without exogenously added saturated fats, high levels ofexogenously-added emulsifiers and/or other types of thickeners. Suchcompositions and methods to form such compositions would be highlydesirable. It would be further desirable to provide a low cost methodfor making such a composition, said method involving the use ofnon-hazardous materials, minimal processing steps, and minimal rawmaterial inventory.

Liquid oils such as, microbial oils, known to contain a high content ofLC-PUFAs are typically processed for consumption by humans or otheranimals by multiple steps, including pretreatment, desolventization ordeodorization, winterization, caustic refining (also known as chemicalrefining), chill filtration, and bleaching. Such processes add time andcost to preparation of products and can introduce chemicals in therefining process unacceptable for the natural or organic productsmarket. Accordingly, there is a need for improved methods of producingoils that are simplified, less costly and acceptable to broad markets,while still being effective for producing products having acceptableorganoleptic properties.

SUMMARY OF THE INVENTION

The present invention provides a method for producing a solid fatcomposition comprising mixing an oil comprising saturated fat and amicrobial oil comprising at least one LC-PUFA with at least oneemulsifier to form a mixture; and solidifying the mixture to form asolid fat composition. The invention also provides a solid fatcomposition comprising a mixture of an unwinterized microbial oilcomprising an LC-PUFA and an emulsifier, wherein the mixture is a solidcomposition at room temperature.

In some embodiments of the method, the oil comprises between about 5 wt.% and about 70 wt. % LC-PUFA and between about 20 wt. % and about 60 wt.% saturated fat.

In some embodiments, the solid fat composition comprises saturated fat.

In some embodiments, the saturated fat is not added exogenously, and inother embodiments, the saturated fat is added exogenously. In furtherembodiments, the microbial oil is unwinterized or not hydrogenated.

In some embodiments, the microbial oil is from a microorganism selectedfrom the group consisting of microorganisms of the genusThraustochytrium, microorganisms of the genus Schizochytrium,microorganisms of the genus Althornia, microorganisms of the genusAplanochytrium, microorganisms of the genus Japonochytrium,microorganisms of the genus Labyrinthula, microorganisms of the genusLabyrinthuloides, microorganisms of the genus Crypthecodinium, andmixtures thereof. In further embodiments, the microorganism is selectedfrom the group consisting of microorganisms of the genusThraustochytrium, microorganisms of the genus Schizochytrium,microorganisms of the genus Crypthecodinium, and mixtures thereof.

In some embodiments, the microbial oil comprises an LC-PUFA having acarbon chain length of at least 20, or at least 22, or has at leastthree double bonds, or has at least four double bonds. In someembodiments, the LC-PUFA comprises docosahexaenoic acid, ordocosapentaenoic acid, or arachidonic acid, or eicosapentaenoic acid. Inother embodiments, the oil comprises at least about 50 weight percentdocosahexaenoic acid, or at least about 60 weight percentdocosahexaenoic acid.

In some embodiments, the solid fat composition has a homogeneoustexture, or is a shortening.

In some embodiments, the emulsifier is a monoglyceride, a diglyceride, amono/diglyceride combination, a lecithin, a lactylated mono-diglyceride,a polyglycerol ester, a sucrose fatty acid ester, a sodium steroyllactylate, a calcium steroyl lactylate, or combinations thereof. Infurther embodiments, the emulsifier is present in an amount of betweenabout 0.01 weight percent and about 2.0 weight percent, and in furtherembodiments, between about 0.05 weight percent about 0.2 weight percent.

In some embodiments of the method, the solid fat composition has amelting temperature of at least about 20° C., at least about 30° C., orat least about 35° C.

In some embodiments of the method, the step of solidifying the mixturecontrols formation of crystals in the solid fat composition. Inembodiments of the solid fat composition, the composition comprisescrystals, and in some embodiments, the crystals comprise β-primecrystals. In further embodiments of the method or the solid fatcomposition, the crystals comprise β-prime crystals, at least about 50wt. % of the fats and/or oils in the solid fat composition are in theβ-prime crystal form, or at least about 80 wt. % of the fats and/or oilsin the solid fat composition are in the β-prime crystal form.

In some embodiments of the method, the oil and/or emulsifier is heated,heated prior to the mixing step, or heated to at least about 40° C.

In some embodiments of the method, the mixing step comprises agitatingthe mixture, and in further embodiments, the step of agitating forms acontinuous mixture.

In some embodiments of the method, the step of solidifying the mixturecomprises cooling the mixture, and in further embodiments, the step ofcooling comprises cooling the mixture to a temperature of about 0° C. toabout 3° C., or the step of solidifying further comprises mixing themixture during the step of cooling, or the mixture is cooled at a rateof between about 1° C./min and about 20° C./min.

In some embodiments of the method, the step of solidifying comprisesintroducing nitrogen into the mixture, and can comprise bubblingnitrogen through the mixture.

The method can further comprise adding at least one additionalingredient to the mixture, including a water-soluble liquid, includingwater. The water-soluble liquid can be added at an amount between about1 wt. % and about 10 wt. %.

The composition can further comprise at least one additional ingredient,including a water-soluble liquid, including water. The water-solubleliquid can be present in an amount between about 1 wt. % and about 10wt. %.

The additional ingredient can also be antioxidants, flavors, flavorenhancers, sweeteners, pigments, vitamins, minerals, pre-bioticcompounds, pro-biotic compounds, therapeutic ingredients, medicinalingredients, functional food ingredients, processing ingredients, orcombinations thereof.

In some embodiments, the additional ingredient is ascorbic acid or asalt of ascorbic acid, and in some embodiments is added in an amountbetween about 0.5 wt. % and about 5 wt. %.

In some embodiments, the additional ingredient is an antioxidant, and insome embodiments is ascorbyl palmitate, tocopherols, citric acid,ascorbic acid, tertiary butyl hydroquinone, rosemary extract, lecithin,or mixtures thereof.

In some embodiments, the solid fat composition has an OSI value of atleast about 20, at least about 40, or at least about 60.

In some embodiments of the method, the solid fat composition is selectedfrom the group consisting of a food product, a nutritional product and apharmaceutical product.

In some embodiments of the method, the method further comprises addingthe solid fat composition to a product selected from the groupconsisting of a food product, a nutritional product and a pharmaceuticalproduct.

The present invention also provides a fat composition comprising anunwinterized microbial oil comprising between about 5 wt. % and about 70wt. % LC-PUFA and between about 20 wt. % and about 60 wt. % saturatedfat; and between about 0.01 wt. % and about 2.0 wt. % of an emulsifier,wherein the composition comprises less than about 10 wt. % of water andwherein the composition is a solid composition at room temperature.

In an additional embodiment, the invention provides a method ofpreparing an oil product that is used for consumption, comprisingextracting an oil-containing fraction from a microbial biomass, whereinthe oil-containing fraction comprises at least one LC-PUFA and saturatedfatty acids at least sufficient to visually affect the oil-containingfraction; and treating the oil-containing fraction by vacuum evaporationto produce an oil product comprising at least one LC-PUFA, wherein theoil product has not been subject to a winterization step. The presentinvention also provides an oil product produced by the method.

The invention also provides a microbial oil product that is used forconsumption prepared by extracting an oil-containing fraction from amicrobial biomass, wherein the oil-containing fraction comprises atleast one LC-PUFA and saturated fatty acids at least sufficient tovisually affect the oil-containing fraction, and treating the fractionby a process of vacuum evaporation, wherein the oil product is notsubjected to a winterization step.

In some embodiments of the method, the oil product has not been subjectto a caustic refining process. In other embodiments, the oil product hasnot been subject to a chill filtration process, and in otherembodiments, the oil product has not been subject to a bleachingprocess.

In some embodiments of the method, the oil-containing fraction cancomprise an LC-PUFA having a carbon chain length of at least 20, atleast 22, having at least three double bonds, or at least four doublebonds. In some embodiments, the LC-PUFA can comprise docosahexaenoicacid, docosapentaenoic acid, arachidonic acid, or eicosapentaenoic acid.

In some embodiments of the method, the step of treating theoil-containing fraction comprises desolventization. In furtherembodiments, the desolventization can comprise subjecting the extractedoil-containing fraction to vacuum conditions at high temperature,including, but not limited to temperatures from about 50° C. to about70° C. The desolventization can also comprise subjecting the extractedoil-containing fraction to a vacuum of greater than a vacuum of about100 mm Hg, subjecting the extracted oil-containing fraction to a vacuumof greater than a vacuum of about 70 mm Hg, or subjecting the extractedoil-containing fraction to a vacuum of greater than a vacuum of about 50mm Hg.

In some embodiments of the method, the step of treating theoil-containing fraction comprises deodorization. In further embodiments,the deodorization comprises subjecting the extracted oil-containingfraction to vacuum conditions at high temperature while sparging theextracted oil-containing fraction with steam. In one aspect, the hightemperature is from about 190° C. to about 220° C. In this embodiment,the desolventization can comprise subjecting the extractedoil-containing fraction to a vacuum of greater than a vacuum of about 25mm Hg, subjecting the extracted oil-containing fraction to a vacuum ofgreater than a vacuum of about 12 mm Hg, or subjecting the extractedoil-containing fraction to a vacuum of greater than a vacuum of about 6mm Hg.

In some embodiments of the method, the oil product has been subjected tothe step of bleaching either before or after the step of treating. Inother embodiments, the method further comprises fractionating the oilinto an olein fraction and a stearin fraction. In other embodiments, theoil product is used for human consumption.

In some embodiments, the oil product has not been subject to a causticrefining process, a chill filtration process, or a bleaching process.

In some embodiments, the microbial biomass is from a microorganismselected from the group consisting of microorganisms of the genusThraustochytrium, microorganisms of the genus Schizochytrium,microorganisms of the genus Althornia, microorganisms of the genusAplanochytrium, microorganisms of the genus Japonochytrium,microorganisms of the genus Labyrinthula, microorganisms of the genusLabyrinthuloides, microorganisms of the genus Crypthecodinium, andmixtures thereof. In other embodiments, the microbial biomass is from amicroorganism selected from the group consisting of microorganisms ofthe genus Schizochytrium, microorganisms of the genus Crypthecodinium,and mixtures thereof.

In some embodiments, the oil product has a free fatty acid content ofless than about 0.5 wt. %, and in other embodiments, has a free fattyacid content of less than about 0.3 wt. %.

In some embodiments, the oil product has a phosphorous value of lessthan about 10 ppm, and in other embodiments, has a phosphorous value ofless than about 5 ppm.

In some embodiments, the oil product has a peroxide value of less thanabout 2 meq/kg, and in other embodiments, a peroxide value of less thanabout 1 meq/kg.

In some embodiments, the oil product has an anisidine value of less thanabout 5, and in other embodiments, has an anisidine value of less thanabout 3.

In some embodiments, the oil product has a soap content of less thanabout 5 wt. %, and in other embodiments, has a soap content of less thanabout 2.5 wt. %.

In some embodiments, the oil product has an Fe concentration of lessthan about 1 ppm, and in other embodiments, has an Fe concentration ofabout 0.5 ppm.

In some embodiments, the oil product has a Pb concentration of less thanabout 1 ppm, and in other embodiments, has a Pb concentration of about0.2 ppm.

In some embodiments, the oil product has an Hg concentration of lessthan about 0.1 ppm, and in other embodiments, has an Hg concentration ofabout 0.04 ppm.

In some embodiments, the oil product has an Ni concentration of lessthan about 0.1 ppm, and in other embodiments, the oil product has an Niconcentration of about 0.01 ppm.

In some embodiments, the oil product has a Cu concentration of less thanabout 1 ppm, and in other embodiments, has a Cu concentration of about0.2 ppm.

The present invention also provides a nutritional product comprising themicrobial oil product, a pharmaceutical product comprising the microbialoil product, and a food product comprising the microbial oil product anda food or liquid component. In some embodiments, the pharmaceuticalproduct further comprises a pharmaceutically acceptable excipient. Inother embodiments, the pharmaceutical product further comprises apharmaceutically active agent selected from the group consisting ofstatins, anti-hypertensive agents, anti-diabetic agents, anti-dementiaagents, anti-depressants, anti-obesity agents, appetite suppressants andagents to enhance memory and/or cognitive function.

In some embodiments, the food product is selected from the groupconsisting of doughs, batters, baked food, liquid food products,semi-solid food products, food bars, processed meats, ice creams, frozendesserts, frozen yoghurts, waffle mixes, salad dressings, replacementegg mixes, salted snacks, specialty snacks, dried fruit snacks, meatsnacks, pork rinds, health food bars, rice/corn cakes, and confectionerysnacks.

In some embodiments, the microbial oil product is used for humanconsumption.

The present invention also provides a microbial oil product that is usedfor consumption prepared by a process, comprising extracting anoil-containing fraction comprising at least one LC-PUFA from a microbialbiomass; and treating the fraction by a process of vacuum evaporation,wherein the oil product is not subjected to a winterization step, acaustic refining process, a chill filtration process, or a bleachingprocess; and wherein the oil product has a characteristic selected fromthe group consisting of a free fatty acid content of less than about 0.5wt. %, a phosphorous value of less than about 10 ppm, a peroxide valueof less than about 2 meq/kg, an anisidine value of less than about 5, asoap content of less than about 5 wt. %, an Fe concentration of lessthan about 1 ppm, a Pb concentration of less than about 1 ppm, an Hgconcentration of less than about 0.1 ppm, an Ni concentration of lessthan about 0.1 ppm, and a Cu concentration of less than about 1 ppm.

Also provided is a food product comprising the microbial oil product anda food or liquid component, a nutritional product comprising themicrobial oil product, and a pharmaceutical product comprising themicrobial oil product.

In some embodiments, the microbial oil product is used for humanconsumption.

The invention also provides a method of preparing an oil product that isused for consumption, comprising extracting an oil-containing fractionfrom a microbial biomass, wherein the oil-containing fraction comprisesat least one LC-PUFA; and treating the oil-containing fraction by vacuumevaporation to produce an oil product comprising at least one LC-PUFA,wherein the oil product has not been subject to a caustic refiningprocess. The present invention also provides an oil product produced bythis method.

In some embodiments, the microorganism is a microorganism of the genusMortierella.

In some embodiments, the oil-containing fraction comprises arachidonicacid.

The invention also provides a blended oil product, comprising: an oilproduct produced a method comprising extracting an oil-containingfraction from a microbial biomass, wherein the oil-containing fractioncomprises at least one LC-PUFA and saturated fatty acids at leastsufficient to visually affect the oil-containing fraction; and treatingthe oil-containing fraction by vacuum evaporation to produce an oilproduct comprising at least one LC-PUFA, wherein the oil product has notbeen subject to a winterization step; and an oil product produced by amethod of comprising extracting an oil-containing fraction from amicrobial biomass, wherein the oil-containing fraction comprises atleast one LC-PUFA, and treating the oil-containing fraction by vacuumevaporation to produce an oil product comprising at least one LC-PUFA,wherein the oil product has not been subject to a caustic refiningprocess.

In some embodiments, the microbial biomass from which the former oilproduct was produced is from a microorganism selected from the groupconsisting of microorganisms of the genus Thraustochytrium,microorganisms of the genus Schizochytrium, microorganisms of the genusAlthornia, microorganisms of the genus Aplanochytrium, microorganisms ofthe genus Japonochytrium, microorganisms of the genus Labyrinthula,microorganisms of the genus Labyrinthuloides, microorganisms of thegenus Crypthecodinium, and mixtures thereof. In further embodiment, themicroorganism is selected from the group consisting of microorganisms ofthe genus Schizochytrium, microorganisms of the genus Crypthecodinium,and mixtures thereof.

In a further embodiment of the blended oil product, the microbialbiomass from which the latter oil product was produced is from amicroorganism of the genus Mortierella.

In a further embodiment, the blended oil product comprisesdocosahexaenoic acid and arachidonic acid.

In any of the embodiments of the present invention, in one aspect, anoil product produced by a process or method of the invention is a solidat 20° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates various alternative embodiments of the presentinvention for producing a PUFA-containing oil of the present invention.

FIG. 2 illustrates various alternative embodiments of the presentinvention for producing a PUFA-containing oil of the present invention.

FIG. 3 illustrates a comparison of the oxidative stability index of asolid fat composition, solid fat composition with added ascorbic acid,and a solid fat composition with added ascorbic acid and folic acid.

DETAILED DESCRIPTION OF THE INVENTION

The food, nutritional, and pharmaceutical product compositions andmethods for preparation of the same, as taught by the present invention,allow for increased intake of nutrients, particularly LC-PUFAs,particularly omega-3 and omega-6 LC-PUFAs, which can provide healthbenefits to those consuming such products. The present invention isdirected in part towards a high-quality PUFA-containing oil productprepared with minimal processing that has improved functionality,improved stability and is compatible with a broad range of applicationsincluding the natural and/or organic market sector. One particularlypreferred use of such oil products is in the production of a solid fatcomposition comprising LC-PUFAs that can be used in, or as a,nutritional product, a food product, and/or a pharmaceutical product(medicinal and/or therapeutic). The oils for making products of theinvention are microbial oils containing LC-PUFAs derived from amicrobial biomass.

A first embodiment of the present invention is a process for producingminimally processed microbial oils that are high-quality PUFA-containingoil products. The process includes extracting an oil-containing fractioncomprising at least one LC-PUFA from a microbial biomass to produce amicrobial oil. Microbial sources and methods for growing microorganismscomprising nutrients and/or LC-PUFAs for recovery in microbial oils areknown in the art (Industrial Microbiology and Biotechnology, 2^(nd)edition, 1999, American Society for Microbiology). Preferably, themicroorganisms are cultured in a fermentation medium in a fermentor. Themethods and compositions of the present invention are applicable to anyindustrial microorganism that produces LC-PUFA.

Microbial sources can include a microorganism such as an algae,bacteria, fungi (including yeast) and/or protist. Preferred organismsinclude those selected from the group consisting of golden algae (suchas microorganisms of the kingdom Stramenopiles), green algae, diatoms,dinoflagellates (such as microorganisms of the order Dinophyceaeincluding members of the genus Crypthecodinium such as, for example,Crypthecodinium cohnii), yeast, and fungi of the genera Mucor andMortierella, including but not limited to Mortierella alpina andMortierella sect. schmuckeri. Members of the microbial groupStramenopiles include microalgae and algae-like microorganisms,including the following groups of microorganisms: Hamatores,Proteromonads, Opalines, Develpayella, Diplophrys, Labrinthulids,Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation,Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus,Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae),Eustigmatophytes, Raphidophytes, Synurids, Axodines (includingRhizochromulinaales, Pedinellales, Dictyochales), Chrysomeridales,Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales. TheThraustochytrids include the genera Schizochytrium (species includeaggregatum, limnaceum, mangrovei, minutum, octosporum), Thraustochytrium(species include arudimentale, aureum, benthicola, globosum, kinnei,motivu, multirudimentale, pachydermum, proliferum, roseum, striatum),Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda,radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis),Aplanochytrium (species include haliotidis, kerguelensis, profunda,stocchinoi), Japonochytrium (species include marinum), Althornia(species include crouchii), and Elina (species include marisalba,sinorifica). The Labrinthulids include the genera Labyrinthula (speciesinclude algeriensis, coenocystis, chattonii, macrocystis, macrocystisatlantica, macrocystis macrocystis, marina, minuta, roscoffensis,valkanovii, vitellina, vitellinapacifica, vitellina vitellina, zopfi),Labyrinthomyxa (species include marina), Labyrinthuloides (speciesinclude haliotidis, yorkensis), Diplophrys (species include archeri),Pyrrhosorus* (species include marinus), Sorodiplophiys* (species includestercorea), Chlamydomyxa* (species include labyrinthuloides, montana).(*=there is no current general consensus on the exact taxonomicplacement of these genera). While processes of the present invention canbe used to produce forms of nutrients that can be produced in a widevariety of microorganisms, for the sake of brevity, convenience andillustration, this detailed description of the invention will discussprocesses for growing microorganisms which are capable of producinglipids comprising omega-3 and/or omega-6 polyunsaturated fatty acids, inparticular microorganisms that are capable of producing DHA, DPA n-3,DPA n-6, EPA or ARA. Additional preferred microorganisms are algae, suchas Thraustochytrids of the order Thraustochytriales, includingThraustochytrium (including Ulkenia) and Schizochytrium, and includingThraustochytriales which are disclosed in commonly assigned U.S. Pat.Nos. 5,340,594 and 5,340,742, both issued to Barclay, all of which areincorporated herein by reference in their entirety. More preferably, themicroorganisms are selected from the group consisting of microorganismshaving the identifying characteristics of ATCC number 20888, ATCC number20889, ATCC number 20890, ATCC number 20891 and ATCC number 20892. Sincethere is some disagreement among experts as to whether Ulkenia is aseparate genus from the genus Thraustochytrium, for the purposes of thisapplication, the genus Thraustochytrium will include Ulkenia. Alsopreferred are strains of strains of Mortierella sect. schmuckeri (e.g.,including microorganisms having the identifying characteristics of ATCC74371) and Mortierella alpina (e.g., including microorganisms having theidentifying characteristics of ATCC 42430). Also preferred are strainsof Crypthecodinium cohnii, including microorganisms having theidentifying characteristics of ATCC Nos. 30021, 30334-30348,30541-30543, 30555-30557, 30571, 30572, 30772-30775, 30812, 40750,50050-50060, and 50297-50300. Also preferred are mutant strains derivedfrom any of the foregoing, and mixtures thereof. Oleaginousmicroorganisms are also preferred. As used herein, “oleaginousmicroorganisms” are defined as microorganisms capable of accumulatinggreater than 20% of the weight of their cells in the form of lipids.Genetically modified microorganisms that produce LC-PUFAs are alsosuitable for the present invention. These can include naturallyLC-PUFA-producing microorganisms that have been genetically modified aswell as microorganisms that do not naturally produce LC-PUFAs but thathave been genetically engineered to do so.

Suitable organisms may be obtained from a number of available sources,including by collection from the natural environment. The American TypeCulture Collection currently lists many publicly available strains ofmicroorganisms identified above. As used herein, any microorganism, orany specific type of organism, includes wild strains, mutants, orrecombinant types. Growth conditions in which to culture these organismsare known in the art, and appropriate growth conditions for at leastsome of these organisms are disclosed in, for example, U.S. Pat. No.5,130,242, U.S. Pat. No. 5,407,957, U.S. Pat. No. 5,397,591, U.S. Pat.No. 5,492,938, U.S. Pat. No. 5,711,983, U.S. Pat. No. 5,882,703, U.S.Pat. No. 6,245,365, and U.S. Pat. No. 6,607,900, all of which areincorporated herein by reference in their entirety.

Microbial oils useful in the present invention can be recovered frommicrobial sources by any suitable means known to those in the art. Forexample, the oils can be recovered by extraction with solvents such aschloroform, hexane, methylene chloride, methanol and the like, or bysupercritical fluid extraction. Alternatively, the oils can be extractedusing extraction techniques, such as are described in U.S. Pat. No.6,750,048 and PCT Patent Application Serial No. US01/01806, both filedJan. 19, 2001, and entitled “Solventless Extraction Process,” both ofwhich are incorporated herein by reference in their entirety. Additionalextraction and/or purification techniques are taught in PCT PatentApplication Serial No. PCT/IB01/00841 entitled “Method for theFractionation of Oil and Polar Lipid-Containing Native Raw Materials”filed Apr. 12, 2001; PCT Patent Application Serial No. PCT/IB01/00963entitled “Method for the Fractionation of Oil and Polar Lipid-ContainingNative Raw Materials Using Water-Soluble Organic Solvent andCentrifugation” filed Apr. 12, 2001; U.S. Provisional Patent ApplicationSer. No. 60/291,484 entitled “Production and Use of a Polar Lipid-RichFraction Containing Stearidonic Acid and Gamma Linolenic Acid from PlantSeeds and Microbes filed May 14, 2001; U.S. Provisional PatentApplication Ser. No. 60/290,899 entitled “Production and Use of aPolar-Lipid Fraction Containing Omega-3 and/or Omega-6 HighlyUnsaturated Fatty Acids from Microbes, Genetically Modified Plant Seedsand Marine Organisms” filed May 14, 2001; U.S. Pat. No. 6,399,803entitled “Process for Separating a Triglyceride Comprising aDocosahexaenoic Acid Residue from a Mixture of Triglycerides” issuedJun. 4, 2002 filed Feb. 17, 2000; and PCT Patent Application Serial No.US01/01010 entitled “Process for Making an Enriched Mixture ofPolyunsaturated Fatty Acid Esters” filed Jan. 11, 2001; all of which areincorporated herein by reference in their entirety. The extracted oilscan be evaporated under reduced pressure to produce a sample ofconcentrated oil material. Processes for the enzyme treatment of biomassfor the recovery of lipids are disclosed in U.S. Provisional PatentApplication No. 60/377,550, entitled “HIGH-QUALITY LIPIDS AND METHODSFOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS,” filed on May 3,2002; PCT Patent Application Serial No. PCT/US03/14177 entitled“HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING BY ENZYMATIC LIBERATIONFROM BIOMASS,” filed on May 5, 2003; copending U.S. patent applicationSer. No. 10/971,723, entitled “HIGH-QUALITY LIPIDS AND METHODS FORPRODUCING BY LIBERATION FROM BIOMASS,” filed on Oct. 22, 2004; EP PatentPublication 0 776 356 and U.S. Pat. No. 5,928,696, both entitled“Process for extracting native products which are not water-soluble fromnative substance mixtures by centrifugal force,” the disclosures ofwhich are hereby incorporated by reference herein in their entirety.

In preferred embodiments, the microbial crude oils of the invention arehigh quality microbial crude oils prepared by processes as describedabove. Such oils of the present invention have significant advantagesover, for example, fish oils that typically provide poor quality crudeoils, e.g., because recovery from fish biomass typically involvescooking and hexane extraction and because the oil can containcontaminants and/or other undesirable components and/or undesirablefatty acid profiles.

The microbial oil-containing fraction comprising at least one LC-PUFA,extracted from a microbial biomass as described above, includes at leastone LC-PUFA (i.e., PUFAs having 20 or more carbons). Preferred PUFAs ofthe present invention include C20, C22, or C24 omega-3 or omega-6 PUFAs.Preferably, the PUFA is a long chain PUFA (LC-PUFA), comprising a C20 orC22 omega-3, or a C20 or C22 omega-6 polyunsaturated fatty acid. AnLC-PUFA of the present invention contains at least two double bonds andpreferably, three double bonds, and even more preferably at least fourdouble bonds. PUFAs having 4 or more unsaturated carbon-carbon bonds arealso commonly referred to as highly unsaturated fatty acids, or HUFAs.In particular, the LC-PUFA can include docosahexaenoic acid (at leastabout 10, about 20, about 30, about 40, about 50, about 60, about 70 orabout 80 weight percent of total fatty acids), docosapentaenoic acid n-3(at least about 10, about 20, about 30, about 40, about 50, about 60,about 70 or about 80 weight percent of total fatty acids),docosapentaenoic acid n-6 (at least about 10, about 20, about 30, about40, about 50, about 60, about 70 or about 80 weight percent of totalfatty acids), arachidonic acid (at least about 10, about 20, about 30,about 40, about 50, about 60, about 70 or about 80 weight percent oftotal fatty acids) and/or eicosapentaenoic acid (at least about 10,about 20, about 30, about 40, about 50, about 60, about 70 or about 80weight percent of total fatty acids). The PUFAs can be in any of thecommon forms found in natural lipids including but not limited totriacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,free fatty acids, esterified fatty acids, or in natural or syntheticderivative forms of these fatty acids (e.g. calcium salts of fattyacids, ethyl esters, etc). In preferred embodiments, the microbialoil-containing fraction comprises at least about 70 wt. % of the PUFAsin the fraction in the triglyceride form, at least about 80 wt. %, atleast about 90 wt. %, and at least about 95 wt. %. The term LC-PUFA, asused in the present invention, can refer to either an oil comprising asingle omega-3 LC-PUFA such as DHA, an oil comprising a single omega-6LC-PUFA such as ARA or DPA n-6, or an oil comprising a mixture of two ormore LC-PUFAs such as DHA, DPA n-6, ARA, and EPA. In preferredembodiments, the product comprises an LC-PUFA in combination with atleast one other nutrient.

In addition to the use of a microbial biomass for the extraction ofoils, the present invention also includes the use of oil seeds as abiomass for extraction or recovery of LC-PUFAs. Such oils extracted froman oil seed biomass can be processed and treated as disclosed herein toproduce oil products. For example, oil seeds from any higher plant, andparticularly consumable plants, including crop plants and especiallyplants used for their oils. Such plants can include, for example:canola, soybeans, rapeseed, linseed, corn, safflowers, sunflowers andtobacco. Other preferred plants include those plants that are known toproduce compounds used as pharmaceutical agents, flavoring agents,nutraceutical agents, functional food ingredients or cosmetically activeagents or plants that are genetically engineered to produce thesecompounds/agents. Particularly preferred plants include plants that havebeen genetically modified to produce LC-PUFAs, such as plants into whichgenes for a polyketide synthase system have been introduced. Forexample, such genes and methods of plant transformation are disclosed inPCT Publication No. WO 02/083870 A2, PCT Publication No. WO 2004/087879A2, PCT Publication No. WO 2000/42195 A2, US Patent Publication No.US-2005-0100995-A1, U.S. Provisional Patent Application Ser. No.60/671,656, filed on Apr. 15, 2005, and US Patent Publication No.US-2005-0014231-A1, all of which are incorporated herein by reference.

Such seeds are treated by conventional methods to recover oils, such asby cleaning, dehulling and grinding. The seeds can then be pressed toproduce an oil or contacted with a solvent, such as after flaking, toextract an oil. Suitable solvents can include organic solvents, watermiscible solvents and water. A preferred solvent is hexane.

A further characteristic of PUFA-containing oil products in variousembodiments of the invention is that they contain saturated fatty acidsthat are at least sufficient to visually affect the oil-containingfraction. Many PUFA-containing oil products contain sufficient amountsof saturated fatty acids in forms that, at room temperature (i.e., 20°C.), visually affect the oil, such as by causing cloudiness in the oil.Some such products are even paste-like due to the presence of saturatedfatty acids, for example because they contain sufficient saturated fattyacids in the form of triglycerides. While in conventional processing,such oil products are winterized to remove the saturated fatty acids,the present invention recognizes that commercially valuable products canbe prepared from such oil products without winterization as discussed inmore detail below.

In preferred embodiments of the present invention, oils have a lipidprofile particularly suitable for producing solid or semi-solidcompositions comprising LC PUFAs. More particularly, such oils arerelatively concentrated in highly unsaturated compounds (e.g., 4, 5 orhigher points of unsaturation), relatively concentrated in saturatedcompounds, and/or relatively unconcentrated in mono-, di-, andtri-saturated compounds. Such compositions can be characterized ashaving a bimodal distribution of compounds in terms of saturation, i.e.,high amounts of saturated compounds and high amounts of highlyunsaturated compounds, with low amounts of compounds with intermediateamounts of unsaturatation. For example, such oils can have greater thanabout 20% by weight, greater than about 25% by weight, greater thanabout 30% by weight, greater than about 35% by weight, greater thanabout 40% by weight, greater than about 45% by weight, or greater thanabout 50% by weight of highly unsaturated compounds having 4 or morepoints of unsaturation. In other embodiments, such oils can have greaterthan about 20% by weight, greater than about 25% by weight, greater thanabout 30% by weight, greater than about 35% by weight, greater thanabout 40% by weight, greater than about 45% by weight, or greater thanabout 50% by weight of highly unsaturated compounds having 5 or morepoints of unsaturation. Alternatively or in addition, such oils can havegreater than about 30% by weight, greater than about 35% by weight,greater than about 40% by weight, greater than about 45% by weight, orgreater than about 50% by weight of saturated compounds. Alternativelyor in addition, such oils can have less than about 25% by weight, lessthan about 20% by weight, less than about 15% by weight, less than about10% by weight, or less than about 5% by weight of mono-, di- ortri-saturated compounds.

A process of the invention for producing minimally processedhigh-quality PUFA-containing oil products comprising at least oneLC-PUFA further includes treating the extracted oil-containing fractionproduced as described above. Such further treatment includes a processof vacuum evaporation to produce an oil product comprising at least oneLC-PUFA.

The process of desolventization or drying by high vacuum evaporation isgenerally known in the art and includes subjecting an extracted oil tovacuum conditions, preferably at high temperatures (e.g., from about 50°C. to about 70° C.). For example, the oil can be subjected to a vacuumof greater than a vacuum of about 100 mm Hg, greater than a vacuum ofabout 70 mm Hg, and greater than a vacuum of about 50 mm Hg. As usedherein, for example, reference to “to a vacuum of greater than a vacuumof about 100 mm Hg” means a stronger vacuum such as, e.g., a vacuum of90 mm Hg or 80 mm Hg. Under these conditions, any solvents, water orother components in the extracted oil having a boiling point below theoil will be driven off.

The process of deodorization is generally known in the art and includessubjecting an extracted oil to vacuum conditions to remove any lowmolecular weight components that may be present. Typically, thesecomponents are removed by sparging with steam at high temperatures,under high vacuum. For example, the oil is generally subjected tovacuums greater than those noted above for desolventization.Specifically, the vacuum can be a vacuum of greater than a vacuum ofabout 50 mm Hg, greater than a vacuum of about 25 mm Hg, greater than avacuum of about 12 mm Hg, greater than a vacuum of about 6 mm Hg, andtypically can be between a vacuum of about 12 mm Hg and a vacuum ofabout 6 mm Hg or be between a vacuum of about 6 mm Hg and a vacuum ofabout 1 mm Hg. This process also destroys many peroxide bonds that maybe present and reduces or removes off odors and helps improve thestability of the oil. In addition, under these conditions, solvents,water or other components in the extracted oil having a boiling pointbelow the oil will be driven off. Deoderization is typically performedat high temperatures, such as temperatures between about 190° C. andabout 220° C.

The oil product resulting from this process is a high-qualityPUFA-containing oil that is used for or suitable for consumption byhumans and non-human animals. That is the organoleptic properties of theoil are such that consumption of the product is acceptable to humans andnon-human animals. Specifically, the oil product can contain lowconcentrations of free fatty acids, phosphorous, peroxide values,anisidine values, soaps and heavy metals. Production of this oil by thepresent invention minimizes the amount of downstream processing requiredto bring a microbial oil to acceptable commercial conditions. Specificmodifications include the elimination of a solvent winterization step,the elimination of a caustic refining process, the elimination of achill filtration process, and the possible elimination of a bleachingprocess. In addition, a high-vacuum evaporation process can besubstituted for a deodorization process. The foregoing processdescription facilitates the production of a solid or semi-solid productby retaining the presence of sufficient saturated compounds to preventthe composition from being liquid at room temperature (i.e., about 20°C.). The invention allows production of edible oils from crude microbialoils with exceptionally high recoveries (95-100%) that are compatiblewith the natural and/or organic market sector.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have lowconcentrations of free fatty acids. Measurement of concentrations offree fatty acids of oils is well known in the art. More particularly,oils of the invention can have a free fatty acid content of less thanabout 0.5 wt. %, less than about 0.1 wt. %, and less than about 0.05 wt.%.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have low phosphorousvalues. Measurement of phosphorous values of oils is well known in theart. More particularly, oils of the invention can have a phosphorousvalue of less than about 10 ppm, less than about 5 ppm, and about 0 ppm.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have low peroxidevalues. Measurement of peroxide values of oils is well known in the art.More particularly, oils of the invention can have an peroxide value ofless than about 2 meq/kg, less than about 1 meq/kg, and about 0 meq/kg.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have low anisidinevalues. Measurement of anisidine values of oils is well known in theart. More particularly, oils of the invention can have an anisidinevalue of less than about 5, less than about 3, less than about 2, lessthan about 1, less than about 0.5, less than about 0.3, less than about0.1, and below detection.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have lowconcentrations of soaps. Measurement of concentrations of soap of oilsis well known in the art. More particularly, oils of the invention canhave soap contents of less than about 5 wt. %, less than about 2.5 wt.%, and of 0 wt. %.

In various embodiments, oil products of the present invention, such asoils produced without being subjected to one or more of the conventionalprocessing steps of solvent winterization, caustic refining process,chill filtration process, and a bleaching process, have low heavy metalvalues. Measurement of heavy metal values of oils is well known in theart. More particularly, oils of the invention can have Fe concentrationsof less than about 1 ppm, less than about 0.5 ppm, and preferably atabout 0 ppm; Pb concentrations of less than about 1 ppm, less than about0.2 ppm, and preferably at about 0 ppm; Hg concentrations of less thanabout 0.1 ppm, less than about 0.04 ppm, and preferably at about 0 ppm;Ni concentrations of less than about 0.1 ppm, less than about 0.01 ppm,and preferably at about 0 ppm; Cu concentrations of less than about 1ppm, less than about 0.2 ppm, and preferably at about 0 ppm.

Processes of the present invention to produce minimally processedhigh-quality PUFA-containing oil products having at least one LC-PUFAcan optionally include a step of bleaching the oil product either beforeor after the step of deodorization or the step of high vacuumfractionation, although it is more commonly conducted before the step ofdeodorization. Bleaching of oils is well known in the art and can beaccomplished in conventional processes. Specifically, for example, asilica adsorbent (such as, Trysil 600 (Grace Chemicals)) for removingremnant soap and a bleaching clay can be introduced to the oil and thenfiltered out. Typically, the silica adsorbent is added before thebleaching clay.

Processes of the present invention to produce high-qualityPUFA-containing oil products having at least one LC-PUFA can include aprocess to produce a liquid LC PUFA-containing oil fraction and an LCPUFA-containing solid fat product. Such a process includes a step offractionating a high quality microbial crude oil, as disclosed herein,into an oil product and related solid fat product. Such crude oilproducts can be prepared by extracting an oil-containing fractioncontaining at least one LC-PUFA and saturated fatty acids from amicrobial biomass. The oil-containing fraction can be treated bywinterization, chill filtration, vacuum evaporation and/or other meansto produce a liquid oil product comprising at least one LC-PUFA and asolid product comprising at least one LC-PUFA. Such other means caninclude filtration to separate the liquid oil fraction from a solidcomposition.

The solid fraction components (possibly including adsorbents) can berecovered by solid/liquid separation techniques. Any adsorbents can beseparated from the solid fraction by heating the adsorbents and solidfat material to melt the solid fat material. The adsorbents can then beseparated from the melted solids, by filtering, for example, and themelted solids can then be resolidified by cooling.

The recovered solid fraction will contain a high level of LC PUFA. Inpreferred embodiments, the solid fraction will comprise at least about20%, at least about 25%, at least about 30% by weight LC PUFA and inparticular DHA. Each of the clear oil and the solid can be used as afood or food additive, for example.

Oil products produced in accordance the present invention can be a solidor semi solid materials. As used herein, the term “oil” will includethose materials that are solid or semi solid at room temperature, aswell as those materials that are liquid at room temperature.

Processes of the present invention to produce minimally processedhigh-quality PUFA-containing oil products having at least one LC-PUFAcan optionally include a step of fractionating the oil into an oleinfraction and a stearin fraction after either the step of deodorizationor the step of high vacuum fractionation. Fractionation of oils intoolein and stearin fractions can be applied to any crude, or bleached ordeodorized oil to produce a clear olein fraction and a hard stearinfraction. Due to differences in their physical properties, olein andstearin can be used in different food applications. In conventionalprocesses, stearin is a byproduct of miscella winterization and chillfiltration and is disposed of resulting in ˜30% losses. Fractionationallows production of a saleable stearin fraction. An example of thisfractionation is shown below in Example 5.

With reference to FIG. 1, various alternative embodiments of the presentinvention are illustrated. A starting material, such as a biomass, suchas a spray dried biomass can be subjected to treatment by a solvent forextraction of a crude oil. Such crude oils will include long chainpolyunsaturated fatty acids. The crude oil can be subjects to highvacuum evaporation which will remove extraction solvents, water andother components in the crude oil having a lower boiling point than thedesired oil components. Alternatively, the crude oil can be subjected toan optional bleaching step, such as to remove carotenoids. Theoptionally treated crude oil is then subjected to deodorization bysparging the oil with steam at high temperatures, under high vacuum. Thefinal oil product produced by either the high vacuum evaporation or thedeodorization can then be optionally treated by fractionation into anolein fraction and a stearin fraction.

With reference to FIG. 2, various alternative embodiments of the presentinvention are illustrated by a flow sheet. In its most basic form, theprocess must include the steps of starting with a pasteurizedfermentation broth containing a microbial biomass. The broth ispretreated to release oil from the cells by lysing, such as by enzymatictreatment or mechanical disruption. The pretreated fermentation broth isthen subjected to an extraction step to produce a microbial oil. At aminimum, the process then includes a deodorization step as describedherein. In one alternative process, the process includes a bleachingstep by which the extracted microbial oil is subjected to bleachingprior to the step of deodorization. In further alternative embodiments,winterization steps (i.e., chill filtration) can be conducted on theextracted microbial oil prior to the step of bleaching and/or betweenthe step of bleaching and deodorization.

Processes for producing minimally processed oils of the presentinvention and the resulting products have a number of significantadvantages. Compared to conventional methods of producingPUFA-containing oil products, the present invention has a lower cost,reduced processing requirements, increased manufacturing throughput,increased safety of the processing steps, and eliminates waste/byproductstreams. Moreover, the current process is consistent with the naturaland/or organic market sector. Conventional methods of oil processingtypically utilize all facets of downstream processing, includingchemical refining. Physical refining methods (i.e., methods that do notinvolve caustic refining) have not been extended to fish oil and similarPUFA-containing oils, possibly because of the known difficulties in theprocessing of such oils. Moreover, many of the known physical processingmethods or less refined products are limited because of odor and tastelimitations. Surprisingly, the process of the invention produces bettertasting oils using physical methods and minimum steps.

As described more fully below, the high quality PUFA-containing oilproducts of the present invention can be used in a variety of foodproducts and applications. The oil products can be consumed directly byhumans as a nutritional, dietary, medicinal, or pharmaceutical product.In addition, the oil products can be combined with any known human foodor liquid for consumption by humans to improve nutrition. The oilproducts can also be fed to animals directly as a feed or as asupplement to animal feed. In this manner, any animal-based foodproducts can have enhanced quality when consumed by humans.

In one embodiment, the oil products of the present invention can be usedto supplement infant formula. Infant formula can be supplemented with,for example, a physically refined oil derived from an ARA-producingmicroorganism such as Mortierella alpina or Mortierella sect.schmuckeri, either alone or in combination with other oils such as fishoil or additional oils rich in DHA, such as microbial oils, includingDHA-S™ and DHA-T™ oils (Martek Biosciences, Columbia, Md.). Suchphysically refined ARA-containing oils would not have been chemicallyrefined. Alternatively, infant formula can be supplemented with, forexample, a minimally processed oil derived from a DHA-producingmicroorganism, such as Crypthecodinium cohnii, either alone or incombination with other oils rich in ARA including ARASCO® (MartekBiosciences, Columbia, Md.). In an additional embodiment, infant formulacan be supplemented with multiple oils of the present invention that arederived from more than one source such as, for example, a minimallyprocessed oil containing DHA (e.g., from Crypthecodinium cohnii) and aphysically refined oil containing ARA (e.g., from Mortierella alpina).

In other embodiments, the oil products of the present invention can becombined to produce a blend. For example, a minimally processed oil fromCrypthecodinium cohnii can be blended with a physically refined oil fromMortierella alpina and the resulting blend can be used to supplementinfant formula. Blends of ARA-containing oils and DHA-containing oilsusing oils of the present invention can be produced in a variety ofdifferent ratios of ARA to DHA. Such blends can include ratios ofARA:DHA from about 1:1 to about 2:1. More particularly, the blends canbe produced having ARA:DHA ratios of about 1:1, 1.25:1, 1.5:1, 1.75:1 or2:1.

In a particularly preferred embodiment, the high quality PUFA-containingoil products of the present invention can be used as a starting materialfor the solid fat compositions that are described in detail below. Itshould be appreciated, however, that use of the minimally processed oilproducts of the invention is not limited to a starting material for thesolid fat composition that is described herein.

The inventors have surprisingly discovered that in preferred embodimentsof the solid fat composition of the present invention, an unwinterizedform of an LC-PUFA rich oil, including an unwinterizedmicrobially-derived docosahexaenoic acid-containing oil (DHA oil), canbe used as a starting material for the solid fat compositions of thepresent invention. The processes for making such compositions therebycan avoid the need for hydrogenation of oils, mixing these oils withhard or saturated fats, or other thickening-type agents. Typically,refined oils, i.e., liquid fish oils or microbial oils are produced asan initial crude oil that is then subjected to refining (which removesphospholipids and free fatty acids) and bleaching (to remove pigments)steps. The oil is then typically winterized to remove saturated fats.

The inventors have surprisingly found that, for example, an unwinterizedmicrobial oil, i.e., where the winterization step is not performed,provides a starting material that does not require the treatments taughtin the prior art to form a solid composition. In addition, unwinterizedoil seed oils, as described above, can be used as an alternative tomicrobial oils as described below. Without being bound by theory, theinventors believe that the saturated fats present in the unwinterizedoil gives a more solid consistency to the oil (as compared to winterizedliquid oil). The methods of the present invention for producing a solidfat composition also overcome the tendency of an unwinterized oil toappear grainy (due to the crystallization of triglycerides) causing suchunwinterized oils to appear like a thick liquid oil with particles. Uponstanding at room temperature, unwinterized oil separates, giving aproduct that appears as a thick liquid oil with solids in it. Thepresent invention can overcome this characteristic of unwinterized oil.Processes of the present invention, produce a smooth product of uniformappearance that is stable (with no apparent separation) when leftstanding at room temperature. The resulting product can have theconsistency of shortening.

In a further embodiment, the present invention includes a method forproducing a solid fat composition. The method includes the step ofmixing an oil that includes saturated fat and a microbial oil with atleast one LC-PUFA with at least one emulsifier to form a mixture. Themixture is then solidified to form a solid fat composition.

A “solid fat composition” refers a composition that is solid, orsemi-solid, at room temperature (i.e., 20° C.). Physicochemicalproperties of fats and oils include their viscosity and meltingtemperature. Preferably, a solid fat composition will have a meltingtemperature of at least about 20° C., at least about 25° C., at leastabout 30° C. and preferably at least about 35° C. Melting temperatureswill vary in their sharpness depending on the number of differentchemical entities that are present. Typically, a mixture of severaltriglycerides has a lower melting point than would be predicted based onthe melting points of the individual triglycerides. The mixture willalso have a broader melting range than that of its individualcomponents. Monoglycerides and diglycerides have higher melting pointsthan triglycerides of similar fatty acid composition. In preferredembodiments, the solid fat composition will remain soft enough to spreadonto food products. Preferably, at room temperatures, the compositionwill be viscous, have retarded flow properties, and/or be more adherentto surfaces than the starting materials from which the product is made.

The oil used in methods of the invention to produce a solid fatcomposition includes a microbial oil with at least one LC-PUFA.Microbial sources and methods for growing microorganisms comprisingnutrients and/or LC-PUFAs for recovery in microbial oils are known inthe art as described in detail above in the description of the minimallyprocessed oils of the present invention. Such microbial sources andmethods are suitable as well for producing microbial oils as a startingmaterial for the solid fat compositions of the present invention.Indeed, minimally processed oils as described above are a preferredstarting material for production of solid fat compositions. It should beappreciated, however, that a wide variety of other microbial oilstarting materials, as described below, can be used as startingmaterials for solid fat compositions of the present invention. In oneparticularly preferred embodiment, the microbial oil is an oil producedaccording to the disclosures in PCT Patent Application Serial No.PCT/IB01/00841 entitled “Method for the Fractionation of Oil and PolarLipid-Containing Native Raw Materials” filed Apr. 12, 2001, published asWO 01/76715 and PCT Patent Application Serial No. PCT/IB01/00963entitled “Method for the Fractionation of Oil and Polar Lipid-ContainingNative Raw Materials Using Water-Soluble Organic Solvent andCentrifugation” filed Apr. 12, 2001, published as WO 01/76385.Disclosures in these two PCT applications describe a microbial oilrecovery process that can be generally referred to as the Friolexprocess.

The microbial oil of the invention includes at least one LC-PUFA (i.e.,PUFAs having 20 or more carbons). Preferred PUFAs of the presentinvention include C20, C22, or C24 omega-3 or omega-6 PUFAs. Preferably,the PUFA is a long chain PUFA (LC-PUFA), comprising a C20 or C22omega-3, or a C20 or C22 omega-6 polyunsaturated fatty acid. An LC-PUFAof the present invention contains at least two double bonds andpreferably, three double bonds, and even more preferably at least fourdouble bonds. PUFAs having 4 or more unsaturated carbon-carbon bonds arealso commonly referred to as highly unsaturated fatty acids, or HUFAs.In particular, the LC-PUFA can include docosahexaenoic acid (at leastabout 10, about 20, about 30, about 40, about 50, about 60, about 70 orabout 80 weight percent of total fatty acids), docosapentaenoic acid n-3(at least about 10, about 20, about 30, about 40, about 50, about 60,about 70 or about 80 weight percent of total fatty acids),docosapentaenoic acid n-6 (at least about 10, about 20, about 30, about40, about 50, about 60, about 70 or about 80 weight percent of totalfatty acids), arachidonic acid (at least about 10, about 20, about 30,about 40, about 50, about 60, about 70 or about 80 weight percent oftotal fatty acids) and/or eicosapentaenoic acid (at least about 10,about 20, about 30, about 40, about 50, about 60, about 70 or about 80weight percent of total fatty acids). The PUFAs can be in any of thecommon forms found in natural lipids including but not limited totriacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,free fatty acids, esterified fatty acids, or in natural or syntheticderivative forms of these fatty acids (e.g. calcium salts of fattyacids, ethyl esters, etc). In preferred embodiments, the microbial oilcomprises at least about 70 wt. % of the PUFAs in the oil in thetriglyceride form, at least about 80 wt. %, at least about 90 wt. %, andat least about 95 wt. %. The term LC-PUFA, as used in the presentinvention, can refer to either an oil comprising a single omega-3LC-PUFA such as DHA, an oil comprising a single omega-6 LC-PUFA such asARA or DPA n-6, or an oil comprising a mixture of two or more LC-PUFAssuch as DHA, DPA n-6, ARA, and EPA. In preferred embodiments, theproduct comprises an LC-PUFA in combination with at least one othernutrient.

In preferred embodiments of the invention, the oil used in methods ofthe invention to produce a solid fat composition can include at leastabout 5 wt. %, at least about 10 wt. %, at least about 15 wt. %, atleast about 20 wt. % of LC-PUFA, at least about 25 wt. %, at least about30 wt. %, at least about 35 wt. % of LC-PUFA, at least about 40 wt. %,at least about 45 wt. %, and at least about 50 wt. % of LC-PUFA. Suchembodiments can also have less that about 30 wt. %, less than about 35wt. %, less than about 40 wt. % less than about 45 wt. % LC-PUFA, lessthat about 50 wt. %, less than about 55 wt. %, less than about 60 wt. %,less than about 65 wt. % LC-PUFA, and less than about 70 wt. % LC-PUFA.

The oil used in methods of the invention to produce a solid fatcomposition, in addition to a microbial oil with at least one LC-PUFA,includes saturated fat. Saturated fats will typically have a highermelting point than the LC-PUFA or mixture of LC-PUFAs. Such a saturatedfat can be added to the oil exogenously. Preferred exogenously addedsaturated fats to add include “hard fats” such as partially hydrogenatedvegetable oils, fully hydrogenated oils, partially hydrogenated lards,and non-trans tropical oils. For example, palm oil and palm kernel oiland fractions thereof (palm and palm kernel olein and palm and palmkernel stearin) can be used. When the composition includes anexogenously added fat, the LC-PUFA oil may or may not be winterized. Apreferred amount of exogenously added fat can be determined by one ofskill in the art depending on the degree of solidity and/or viscosity ofthe starting material and the desired degree of solidity and/orviscosity and/or spread consistency desired in the composition.Exogenously added fats can be added in amounts of from about 20 wt. % toabout 60 wt. %, from about 30 wt. % to about 50 wt. %, and from about 35wt. % to about 45 wt. %.

In preferred embodiments, the saturated fat is not added exogenously,but occurs naturally in the microbial oil. For example, microbial oilscomprising LC-PUFAs may be unprocessed oils extracted by any means knownin the art. In such oils, the amount of saturated fats in the microbialoil can be from about 20 wt. % to about 60 wt. %, from about 30 wt. % toabout 50 wt. %, and from about 35 wt. % to about 45 wt. %.

In preferred embodiments of the present invention, the microbial oil isunwinterized (i.e., unfractionated) and will therefore contain saturatedfats. Winterization refers to the process of removing sediment(typically, high melting solid saturated fats) that appears in manyoils, including vegetable oils, at low temperature, most typicallyinvolving the removal of the quantity of crystallized material byfiltration to avoid clouding of the liquid fractions at refrigeratortemperatures. Such techniques include separating oils into two or morefractions with different melting points. The separated liquid and solidfractions exhibit significant differences in physical and chemicalproperties. Suitable techniques are known in the art, and typicallyinclude the following three steps: (i) cooling of the liquid oil tosupersaturation, resulting in the formation of nuclei forcrystallization, (ii) progressive growth of the crystals by gradualcooling, and (iii) separation of the liquid and crystalline phases.These techniques can include, for example, conventional winterization,detergent fractionation and solvent winterization. Conventionalwinterization includes dry fractional crystallization whereintriglycerides with the highest melting temperature preferentiallycrystallize during cooling from the neat liquid or melted fat. Theprinciple of dry fractionation process is based on the cooling of oilunder controlled conditions without the addition of chemicals. Theliquid and solid phases are separated by mechanical means. The principleof detergent fractionation is similar to dry fractionation based on thecooling of oil under controlled conditions without the addition of asolvent. Subsequently, the liquid and solid phases are separated bycentrifugation after an aqueous detergent solution has been added.Solvent (typically acetone) winterization is used to promotetriglyceride crystal formation, because triglycerides at low temperaturegenerally form more stable crystals with solvent than without solvent.In solvent-aided fractionation, either polar or non-polar solvents maybe used to reduce the viscosity of the system during filtration. Thefractions obtained are then freed from the solvent by distillation.Thus, unwinterized microbial oils are those that have not been subjectedto a winterization or fractionation process.

In further preferred embodiments, the microbial oil is not hydrogenatednor partially hydrogenated. Hydrogenation is known in the art, andincludes processes of chemically adding hydrogen gas to a liquid fat inthe presence of a catalyst. This process converts at least some of thedouble bonds of unsaturated fatty acids in the fat molecules to singlebonds thereby increasing the degree of saturation of the fat. The degreeof hydrogenation, that is the total number of double bonds that areconverted, determines the physical and chemical properties of thehydrogenated fat. An oil that has been partially hydrogenated oftenretains a significant degree of unsaturation in its fatty acids.Hydrogenation also results in the conversion of some cis double bonds tothe trans configuration in which one or more double bonds has migratedto a new position in the fatty acid chain. Current studies indicate thattrans-fatty acids may raise total cholesterol and heart disease risk toabout the same extent as saturated fatty acids and are therefore,undesirable in the diet. The present invention allows for the formationof a solid or semi-solid product without the necessity for hydrogenationor partial hydrogenation. The present method includes mixing at leastone emulsifier with the oil including a microbial oil having at leastone LC-PUFA. Preferred emulsifiers to use with the present invention amonoglyceride, a diglyceride, a mono/diglyceride combination, alecithin, a lactylated mono-diglyceride, a polyglycerol ester, a sucrosefatty acid ester, sodium steroyl lactylate, calcium steroyl lactylate,and combinations thereof. In a preferred embodiment, the emulsifier is amono/diglyceride combination. In a preferred embodiment, the emulsifieris present in the mixture in an amount of between about 0.01 weightpercent and about 2.0 weight percent, in an amount of between about0.025 weight percent and about 1.0 weight percent, and in an amount ofbetween about 0.05 weight percent and about 0.2 weight percent. Withoutintending to be bound by theory, it is thought that an emulsifier mayact to provide stability between various components in the mixture tomaintain a homogeneous composition. Lack of stability may result inseparation of oils or separation of the oil and a water phase.Emulsifiers may also provide functional attributes in addition toemulsification, which include aeration, starch and protein complexing,hydration, crystal modification, solubilization, and dispersion.

The physical step of mixing the emulsifier with the oil is conducted inany conventional manner of mixing known in the art. The compositions aremixed to achieve mixing, such as to achieve a homogeneous liquidsolution. For example, it may be necessary to heat the microbial oiland/or the emulsifier, e.g., to at least about 40° C., so that thecompositions are completely liquid and miscible in each other. In apreferred embodiment, the oil is an unwinterized LC-PUFA rich oil and isheated to at least about 40° C. to solubilize all components of the oil.The emulsifier is, in a preferred embodiment, a mixture of mono anddiglyceride emulsifiers that are heated to form a liquid in a separatecontainer from the oil. The melted oil and emulsifier are then mixedtogether by any known method, preferably by agitation to form acontinuous mixture.

The present method also includes solidifying the mixture of the oil andthe emulsifier to form a solid fat composition. For example, in apreferred embodiment in which the mixture is above room temperature, themixture can be allowed to cool to room temperature. Alternatively, themixture can be actively cooled to room temperature or for example, belowroom temperature. For example, the composition can be cooled to betweenabout 0° C. to about 3° C. to solidify. During the step of cooling,whether active or passive, the mixture can be mixed or agitated. In thismanner, cooling can be controlled so that uniform cooling is achievedwithout creating a stratified composition. Preferably, such coolingconditions are adjusted in order to allow the crystal structure of thefat (i.e., the manner in which the molecules orient themselves in thesolid stage) to reach desired levels resulting in desired productplasticity, functionality, and stability. In general, β-prime crystalsresult in a smooth, creamy consistency. β crystals are typically larger,coarser and grainier than β-prime crystals, and accordingly, aretypically less desirable. Accordingly, in preferred embodiments, thecooling process is controlled so as to allow triglycerides in themixture to reach stable, β-prime configurations to produce a producthaving a smooth consistency. Methods to cool that allow such preferredcrystallization forms include cooling the mixture at a rate of betweenabout 1° C./min and about 20° C./min, between about 5° C./min and about15° C./min, and at about 10° C./min. Without being bound by theory, theinventors believe that some emulsifiers suitable for use with thepresent invention, such as mono and diglycerides, act to at leastpartially influence and/or control triglyceride crystallization in thecomposition to result in β-prime crystals. Preferably, at least about 50wt. % of the fats and/or oils in the solid fat composition, at leastabout 55 wt. %, at least about 60 wt. %, at least about 65 wt. %, atleast about 70 wt. %, at least about 75 wt. %, at least about 80 wt. %,at least about 85 wt. %, at least about 90 wt. %, at least about 95 wt.%, or about 100 wt. % are in the β-prime crystal configuration.

In further embodiments, the step of solidifying the mixture of the oiland the emulsifier to form a solid fat composition can includeintroducing nitrogen through the mixture. For example, nitrogen can bebubbled into the composition. Alternatively, nitrogen can be introducedalong with the emulsion into a low temperature crystallizer.

The introduction of nitrogen during solidification can enhance oxidativestability of the product and can improve the product appearance byproviding a shiny appearance.

In preferred embodiments, the solid fat composition of the presentinvention has a homogeneous texture and therefore, has a uniformappearance and consistency. Another characteristic of these embodimentsis that the composition is stable, and does not separate upon standingor otherwise lose its homogeneous texture, preferably for extendedperiods of time. Thus, the composition does not develop a non-uniformappearance or consistency upon standing. In preferred embodiments, thecomposition of the present invention can stand at least about one day,at least about one week, at least about two weeks, at least about threeweeks, and at least about four weeks at room temperature withoutseparating or otherwise losing its homogeneous texture.

The composition of the present invention can also include a number ofadditional functional ingredients. For example, the compositions of thepresent invention can further include microencapsulants including, forexample, proteins, simple and complex carbohydrates, solids andparticulates. Preferred microencapsulants include cell particulates, gumacacia, maltodextrin, hydrophobically modified starch, polysaccharides,including alginate, carboxymethylcellulose and guar gum,hydrophobically-modified polysaccharides, such as octyl-substitutedstarches, proteins, including whey protein isolates, soy proteins, andsodium caseinate, and combinations thereof. In addition, compositions ofthe invention can include surfactants, including for example, anionicagents, cationic agents, nonionic agents, amphoteric agents,water-insoluble emulsifying agents, finely divided particles andnaturally occurring materials. Anionic agents include carboxylic acids,sulfuric esters, alkane sulfonic acids, alkyl aromatic sulfonic acids,miscellaneous anionic hydrophilic groups; cationic agents include aminesalts, ammonium compounds, other nitrogenous bases, non-nitrogenousbases; nonionic agents include an ether linkage to solubilizing group,ester linkage, amide linkage, miscellaneous linkage, multiple linkages;amphoteric agents include amino and carboxy, amino and sulfuric esters,amino and alkane sulfonic acids, amino and aromatic sulfonic acids,miscellaneous combinations of basic and acidic groups; water insolubleemulsifying agents include ionic hydrophilic groups, nonionichydrophilic groups; finely divided particles include any finely dividednon-solubilized particle including clays and carbon; naturally occurringmaterials include alginates, cellulose derivatives water-soluble gums,lipids and sterols, phospholipids, fatty acids, alcohols, proteins,amino acids, detergents; and hydrophilic colloids. Other optionalingredients include thickening agents that include polysaccharides.Thickeners are ingredients that are used to increase the viscosity ofthe composition. In such embodiments, the additional functionalingredient(s) are typically added during the step of mixing.

In one embodiment, the solid fat composition is a shortening.Shortenings typically have little to no added water or aqueous componentand comprise high levels of fats. Alternatively, the solid fatcomposition can be a product such as a margarine, spread, mayonnaise, orsalad dressing. Such products are prepared by blending fats and/or oilswith other ingredients such as water and/or milk products, suitableedible proteins, salt, flavoring and coloring materials and Vitamins Aand D. Margarine typically contains at least 80% fat. Mayonnaise andsalad dressing are semi-solid fatty foods that typically contain notless than 65% and 30% vegetable oil, respectively, and dried whole eggsor egg yolks. Salt, sugar, spices, seasoning, vinegar, lemon juice, andother ingredients complete these products.

Accordingly, the compositions of the present invention can furtherinclude the presence of or the addition of a water-soluble liquid to themixture. Preferably, the water-soluble liquid is water and is added inan amount of less than about 10 wt. %, between about 1 wt. % and about10 wt. %, between about 2 wt. % and about 8 wt. %, and between about 4wt. % and about 6 wt. %. The presence of a water-soluble liquid allowsfor the addition of one or more additional water-soluble ingredients.Any water-soluble ingredient is suitable for the present invention. Apreferred additional ingredient includes antioxidants, flavors, flavorenhancers, sweeteners, pigments, vitamins, minerals, pre-bioticcompounds, pro-biotic compounds, therapeutic ingredients, medicinalingredients, functional food ingredients, processing ingredients, andcombinations thereof.

In a particularly preferred embodiment, the additional ingredient is anantioxidant. Antioxidants are known in the art, and may be added at anypoint in the production of the microbial oil by fermentation or lipidprocessing, or during the processes of the present invention.Antioxidants can help to preserve the resulting products from oxidativedeterioration. Suitable antioxidants may be chosen by the skilledartisan. Preferred antioxidants include ascorbyl palmitate, tocopherols,citric acid, ascorbic acid, tertiary butyl hydroquinone (TBHQ), rosemaryextract, lecithin, and mixtures thereof. Antioxidants can be included inproducts in amounts that are conventional in the art. Particularlypreferred antioxidants include ascorbic acid or a salt of ascorbic acid.In preferred embodiments, when the antioxidant is ascorbic acid or asalt of ascorbic acid, it can be added in amounts up to about 5 wt. %,including amounts ranging from about 0.5 wt. % to about 5 wt. %, fromabout 1.5 wt. % to about 5 wt. %, and from about 3 wt. % to about 5 wt.%. It should be noted that when a water soluble antioxidant, such asascorbic acid, citric acid or salts thereof is added, it must be addedwith water so that it is well dispersed in the composition.Surprisingly, it has been found that the level of increase in theoxidative stability of products of the present invention is greater thanexpected for the amount of antioxidant used, and in particular, when theantioxidant is ascorbic acid or a salt of ascorbic acid. For example,the addition of about 5 wt. % of ascorbic acid or its salt increases theOSI (oxidative stability index) of a composition of the presentinvention three-fold.

The oxidative state and stability of a composition including a lipid maybe measured in a number of ways known in the art, and descriptions ofmany of these techniques are available from the American Oil Chemist'sSociety, as well as from other sources. One method of quantifying theoxidative stability of a product is by measuring the Oxidative StabilityIndex (OSI), such as by use of a Rancimat instrument, that measures theamount of conductive species (volatile decomposition products) that areevolved from a sample as it is subjected to thermal decomposition. Inpreferred embodiments, compositions of the present invention have OSIvalues of at least about 10, at least about 20, at least about 30, atleast about 40, at least about 50, and at least about 60.

In preferred embodiments, the products of the present invention(including the high quality PUFA-containing oil products and the solidfat compositions) are stored under appropriate conditions to minimizeoxidative degradation. Many methods to effect such storage conditionsare known in the art and are suitable for use with the presentinvention, such as, for example, replacement of ambient air with aninert gas atmosphere. A preferred method by which to reduce or minimizeoxidative degradation is to store products under a nitrogen (N₂)atmosphere or mixed nitrogen and carbon dioxide atmosphere. Preferably,packaged products are packaged under nitrogen. Methods for producing anitrogen gas atmosphere into a container comprising a product are knownin the art. In other preferred embodiments, oxidative and/or chemicalstability of this product can also be increased by bubbling nitrogeninto the mixture as it is cooling to provide extra protection againstoxidation.

In another preferred embodiment, products of the present invention cancomprise a pharmaceutically acceptable excipient and/or an addedpharmaceutically active agent (i.e., a therapeutically or medicinallyactive ingredient or combinations thereof). This embodiment isparticularly advantageous for pharmaceutically active agents that havelow solubility in water. Such pharmaceutical products have the advantageof providing therapeutically active ingredients together with beneficialnutrients such as LC-PUFAs. Examples of pharmaceutically acceptableexcipients include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Pharmaceutically active agents ofthe present invention include, without limitation, statins,anti-hypertensive agents, anti-diabetic agents, anti-dementia agents,anti-depressants, anti-obesity agents, appetite suppressants and agentsto enhance memory and/or cognitive function. In another preferredembodiment, products of the present invention can comprise foodingredients such as functional food ingredients, food additives or otheringredients.

The products of the present invention can be used alone as a foodproduct, nutritional product, or pharmaceutical product, or may beincorporated or added to a food, nutritional, or pharmaceutical product.In a first embodiment, the product of the invention is a food productthat includes an oil product of the present invention and a foodcomponent. The products can be used directly as a food ingredient, suchas an oil and/or shortening and/or spread and/or other fatty ingredientin beverages, sauces, dairy-based foods (such as milk, yoghurt, cheeseand ice-cream) and baked goods; or alternately used as a nutritionalproduct, e.g., as a nutritional supplement (in capsule or tablet forms);feed or feed supplement for any animal whose meat or products areconsumed by humans; feed or feed supplement for any companion animal,including without limitation dogs, cats, and horses; food supplement,including baby food and infant formula. The term “animal” means anyorganism belonging to the kingdom Animalia and includes, withoutlimitation, any animal from which poultry meat, seafood, beef, pork orlamb is derived. Seafood is derived from, without limitation, fish,shrimp and shellfish. The term “products” includes any product otherthan meat derived from such animals, including, without limitation,eggs, milk or other products. When fed to such animals, nutrients suchas LC-PUFAs can be incorporated into the flesh, milk, eggs or otherproducts of such animals to increase their content of these nutrients.In addition, when fed to such animals, nutrients such as LC-PUFAs canimprove the overall health of the animal.

The compositions of the present invention can be added to a wide rangeof products such as baked goods, vitamin supplements, diet supplements,powdered drinks, etc. at various stages of production. Numerous finishedor semi-finished powdered food products can be produced using thecompositions of the present invention.

A partial list of food products comprising the products of the presentinvention includes doughs, batters, baked food items including, forexample, such items as cakes, cheesecakes, pies, cupcakes, cookies,bars, breads, rolls, biscuits, muffins, pastries, scones, and croutons;liquid food products, for example, beverages, energy drinks, infantformula, liquid meals, fruit juices, multivitamin syrups, mealreplacers, medicinal foods, and syrups; semi-solid food products such asbaby food, yoghurt, cheese, cereal, pancake mixes; food bars includingenergy bars; processed meats; ice creams; frozen desserts; frozenyoghurts; waffle mixes; salad dressings; and replacement egg mixes. Alsoincluded are baked goods such as cookies, crackers, sweet goods, snackcakes, pies, granola/snack bars, and toaster pastries; salted snackssuch as potato chips, corn chips, tortilla chips, extruded snacks,popcorn, pretzels, potato crisps, and nuts; specialty snacks such asdips, dried fruit snacks, meat snacks, pork rinds, health food bars andrice/corn cakes; and confectionery snacks such as candy.

Another product embodiment of the present invention is a medical food. Amedical food includes a food which is in a formulation to be consumed oradministered externally under the supervision of a physician and whichis intended for the specific dietary management of a disease orcondition for which distinctive nutritional requirements, based onrecognized scientific principles, are established by medical evaluation.

The present invention, while disclosed in terms of specific methods,products, and organisms, is intended to include all such methods,products, and organisms obtainable and useful according to the teachingsdisclosed herein, including all such substitutions, modifications, andoptimizations as would be available to those of ordinary skill in theart. The following examples and test results are provided for thepurposes of illustration and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 Preparation of a High Quality Crude Oil

DHA oil-rich Schizochytrium microorganisms were grown in a fermentor toproduce a fermentation broth. The fermentation broth was harvested andcontacted with Alcalase®2.4, a protease that lysed the Schizochytriumcells. The resulting lysed cell mixture was an emulsion and wascontacted with a 27% solution of isopropanol in water. This mixture wasmixed by agitation and then subjected to centrifugation to produce asubstantially non-emulsified product having two phases. The heavy phasecontained components of the spent fermentation broth, and the lightphase contained DHA-rich oil with some isopropanol and water. The lightphase was dried to produce a high quality crude oil.

Example 2 Minimal Processing of Algal Oil

This example illustrates the production of minimally processed oilsaccording to the present invention.

Minimally processed oils were produced in large scale. Two hundred kg ofhigh quality crude oil (produced as described in Example 1) produced bya Schizochytrium microorganism containing DHA was heated to 65° C. to70° C. under nitrogen. About 0.2% (w/w of oil) of a 50% citric acidsolution was then added into the oil and mixed for 30 to 45 minutesunder nitrogen. Subsequently, 0.2 to 0.5% (w/w of oil) filter aid wasadded into the oil and filtered in order to remove any impuritiespresent in oil. The oil was then deodorized at 210° C. with a feed rateof 180 kg per hour. Deodorized oil was then supplemented withtocopherols, ascorbyl palmitate and rosemary extract. Characteristics ofoils at each process step are given in Table 1. The term “PV” meansperoxide value; the term “FFA” means free fatty acid; and “p-AV” meansp-anisidine value. Recovery from this process was greater than 98%.

TABLE 1 PV FFA Phosphorus DHA Process Step (meq/kg) (%) p-AV (ppm) (%w/w) Crude 0.15 0.22 3.7 3.32 34.0 Citric acid-treated 0.26 0.21 3.6below detection Not analyzed Deodorized 0.28 0.13 4.9 below detectionNot without analyzed antioxidants Deodorized with 0.0 0.15 4.0 belowdetection 33.2 antioxidants

Example 3a Physical Refining

This example illustrates the production of minimally processed oilsaccording to the present invention.

Approximately 600 kg of high quality crude oil (produced as described inExample 1) (FFA <0.3%, Phosphorus <10 ppm, PV <2 meq/kg) was taken andheated to 50-55° C. under nitrogen and/or vacuum. About 0.2% (w/w) of50% citric acid was added and the oil was held at 50-55° C. undernitrogen and/or vacuum for 15 minutes. Trisyl 600 (0.1%-3% w/w, usually0.25%) was added and the temperature was held between 50-55° C. undernitrogen and/or vacuum for 15 minutes. Tonsil Supreme FF bleaching clay(0.1%-4% w/w, usually less than 0.5%) was added and the oil was heatedto 90-95° C. and held under vacuum (>24″ Hg) for 30 minutes. Celite(0.1-0.5% w/w, usually 0.2%) was then added and the oil was filteredthrough a Sparkler filter. The oil was then deodorized at 210-225° C.and a flowrate of 180-225 kg/hr. After deodorization, antioxidants wereadded. This process yielded an oil that is a semi-solid at roomtemperature.

Oil yields from this process ranged from ˜92-97%. Quality data for theseruns with antioxidants are shown in Table 2

TABLE 2 Final Initial Final Initial Final Trial Initial FFA FFA PV PVPhos. Phos. No. (%) (%) (meq/kg) (meq/kg) (ppm) (ppm) Trial #1 <0.1 0.111.15 0 9.2 1.9 Trial #2 <0.1 0.09 0.15 0 5.6 0 Trial #3 0.28 0.19 0.25<0.1 2.6 3.4 Trial #4 0.23 0.21 0.26 0 3.3 0FFAs of deodorized oils were measured before and after antioxidantsaddition. A significant increase in FFAs (about 2×) was observed afteradding antioxidants.

Example 3b Physical Refining (Clear Oil)

This example illustrates the production of minimally processed liquidoils and related solid fat products according to the present invention.

Approximately 1200 kg of high quality crude oil (produced as describedin Example 1) (FFA <0.3%, Phosphorus <12 ppm, PV <2 meq/kg) was takenand heated to 50-55° C. under nitrogen and/or vacuum. About 0.2% (w/w)of 50 wt % citric acid was added and the oil was held at 50-55° C. undernitrogen and/or vacuum for 15 minutes. The oil was then chilled from 55°C. to ˜35° C. under nitrogen and/or vacuum using various hold times(0-12 hrs.) and agitator speeds (4-16 rpm). At this time, celite(0.1-0.5% w/w, usually 0.2%) was added and the oil was filtered througha Sparkler filter. The chill filtration step was repeated with the oilbeing heated under nitrogen and/or vacuum and chilled from ˜50° C. to˜30° C. using various hold times (0-12 hrs.) and agitator speeds (4-16rpm). Celite (0.1-0.5% w/w, usually 0.2%) was added again and the oilwas filtered through a Sparkler filter. Next, Trisyl 600 (0.1%-3% w/w,usually 0.25%) was added and the temperature was held between 50-55° C.under nitrogen and/or vacuum for 15 minutes. Tonsil Supreme FF bleachingclay (0.1%-4% w/w, usually 0.5% or less) was added and the oil washeated to 90-95° C. and held under vacuum (>24″ Hg) for 30 minutes.Celite (0.1-0.5% w/w, usually 0.2%) was added and the oil was filteredthrough a Sparkler filter. The oil was then chilled again under nitrogenand/or vacuum from ˜40° C. to 20° C. using various hold times (0-12hrs.) and agitator speeds (4-16 rpm). Celite (0.1-0.5% w/w, usually0.2%) was added and the oil was filtered through a Sparkler filter. Theoil was then deodorized at 210-225° C. and a flowrate of 180-225 kg/hr.After deodorization, antioxidants were added. This yields an oil that isclear at room temperature. Oil yields from this process range from˜55-60%. Quality data for these runs with antioxidants are shown inTable 3.

TABLE 3 Final Initial Final Initial Final Trial Initial FFA FFA PV PVPhos. Phos. No. (%) (%) (meq/kg) (meq/kg) (ppm) (ppm) Trial #1 0.21 0.10.32 0.5 <5 2.6 Trial #2 0.19 0.17 <0.1 0.07 11 3.1 Trial #3 0.12 0.170.53 0.07 3 6.5 Trial #4 0.18 0.08 0.26 0 3.3 0.5

The material retained by the filter can be treated, for example byheating and filtering, to separate the solid material from the bleachingclay. Heating the material retained by the filter will melt the solids.The melted solids can then be separated from the clay, by filtering, forexample, and then resolidified by cooling. The recovered solid willcontain about 20-30% PUFA, most of which is DHA. The clear oil and thesolid can be used as a food or food additive, for example.

Example 3c Physical refining/Silica Refining

This example illustrates the production of minimally processed oilsaccording to the present invention.

Approximately 100 g of high quality crude oil (produced as described inExample 1) (FFA <0.8%, Phosphorus <10 ppm, PV <2 meq/kg) was taken andheated to 50-55° C. under nitrogen. About 0.2% (w/w) of 50 wt % citricacid was added and the oil was held at 50-55° C. under nitrogen and/orvacuum for 15 minutes. Subsequently, 0.5%-1.25% w/w of silica(Brightsorb F100) was added and the oil was heated to 85° C. undervacuum. After 30 minutes holding time, Tonsil Supreme FF bleaching clay(0.5% w/w) was added, the oil was heated to 90-95° C. and held undervacuum (>24″ Hg) for 30 minutes. Celite (0.1-0.5% w/w, usually 0.2%) wasthen added and the oil was vacuum filtered using a Buchner funnel aftercooling to 60-65° C. Yields for these tests were between 95-96%. Qualityresults for these tests are shown in Table 4. The final product was asemi-solid oil. This product could also be deodorized and/or bleachedand would remain a semi-solid oil.

TABLE 4 Initial Final Initial Final FFA FFA PV PV Initial Final TrialNo. % Silica (%) (%) (meq/kg) (meq/kg) AV AV Trial #1 0.5% 0.64 0.431.51 1.40 6.1 n/a Trial #2 0.8% 0.64 0.34 1.51 1.33 6.1 n/a Trial #31.2% 0.64 0.17 1.51 1.33 6.1 6.3

Example 3d Modified Caustic Refining

This example illustrates the production of minimally processed oilsaccording to the present invention.

Approximately 600 kg of high quality crude oil (produced as described inExample 1) with FFA up to 0.8% (Phosphorus <12 ppm, PV <2 meq/kg) wastaken and heated to 50-55° C. under nitrogen and/or vacuum. About 0.2%(w/w) of 50 wt % citric acid was added and the oil is held at 50-55° C.under nitrogen and/or vacuum for 15 minutes. At this time, 0.1%-0.5% w/wof 50% caustic was added to the oil and held at 60-65° C. for 15-30minutes (this is ˜2-10 times less caustic than the standard amountused). The oil was then centrifuged to remove the soaps from the oil.Trisyl 600 (0.1%-3% w/w, usually 0.25%) was added and the temperaturewas held between 50-55° C. under nitrogen and/or vacuum for 15 minutes.Tonsil Supreme FF bleaching clay (0.1%-4% w/w, usually 0.5% or less) wasadded and the oil was heated to 90-95° C. and held under vacuum (>24″Hg) for 30 minutes. Celite (0.1-0.5% w/w, usually 0.2%) was added andthe oil was filtered through a Sparkler filter. The oil was thendeodorized at 210-225° C. and a flowrate of 180-225 kg/hr. Afterdeodorization, antioxidants were added. This process yielded asemi-solid liquid.

Oil yields from this process range from ˜81-91%. Quality data for theseruns with antioxidants are shown in Table 5.

TABLE 5 Final Initial Final Initial Final Trial Initial FFA FFA PV PVPhos. Phos. No. (%) (%) (meq/kg) (meq/kg) (ppm) (ppm) Trial #1 0.26 <0.11.37 0 11.6 4.0 Trial #2 0.54 <0.1 1.84 0 9.8 4.5 Trial #3 0.75 0.1 0.17<0.1 8.0 5.0 Trial #4 0.40 0.13 0 <0.1 7.0 0.6 Trial #5 0.23 0.08 0.31 03.3 0.9

Example 3e Modified Caustic Refining/No Centrifugation

This example illustrates the production of minimally processed oilsaccording to the present invention.

Approximately 100 g of high quality crude oil (produced as described inExample 1) (FFA <0.3%, Phosphorus <10 ppm, PV <2 meq/kg) was taken andheated to 50-55° C. under nitrogen and/or vacuum. About 0.2% (w/w) of 50wt % citric acid was added and the oil was held at 50-55° C. undernitrogen and/or vacuum for 15 minutes. At this time, 0.4% w/w of 50%caustic was added to the oil and held at 60-65° C. for 15-30 minutes(this is ˜2-10 times less caustic than the standard amount used). Next,Trisyl 600 (1.5% w/w) was added and the temperature was held between50-55° C. under nitrogen and/or vacuum for 15 minutes. Celite (0.2% w/w)was added to the oil and it was vacuum filtered using a Buchner funnel.Tonsil Supreme FF bleaching clay (1.0% w/w) was added to the filteredoil and it was heated to 90-95° C. and held under vacuum (>24″ Hg) for30 minutes. Celite (0.2% w/w) was added and the oil was vacuum filteredusing a Buchner funnel. Quality results for this test are shown in Table6. The final product was a semi-solid oil. This product could also bedeodorized and/or bleached and would remain a semi-solid oil.

TABLE 6 Trial Initial FFA Final FFA Initial PV Final PV Initial FinalNo. (%) (%) (meq/kg) (meq/kg) AV AV Trial #1 0.64 0.14 1.51 1.21 6.1 5.6

Example 4 Dry Fractionation of Crude Algal Oil

This example illustrates the dry fractionation of crude algal oilproduced by a Schizochytrium microorganism containing DHA into olein andstearin fractions according to the present invention.

Three hundred and fifty kg of the crude oil was subjected to the dryfractionation process according to the invention in order to produceliquid olein and solid stearin fractions. Melting of all crystallinephases within the crude algal oil was ensured by heating the same to60-70° C. in a vessel with stirring. The material was then cooledrapidly to 20-30° C. during the pre-cooling phase, with the speed of thestirrer increased to 40 revolutions per minute. In order to obtain thehighest possible heat transfer coefficient in this phase, a liquidcoolant was employed and was water in this example. The temperature ofthe coolant was not permitted to fall significantly below the nucleationtemperature.

The subsequent nucleation phase was conducted within the stirring vesseland was initiated by a reduction of the stirrer speed to 20 revolutionsper minute. Further cooling of the oil was done by regulating thetemperature difference existing between the coolant and the oil, fromthe initial oil temperature of 20-30° C., down to the crystallizationtemperature of about 12-14° C. Once the crystallization temperature hasbeen reached, the stirrer speed was reduced to 15 revolutions perminute. Termination of the crystallization was accomplished bytransferring the suspension into a filtration unit immediately after thedesired cloud point was reached for the remaining oil, the so-calledolein fraction that was still present between the crystals. To monitorthe cloud point of the olein fraction, test filtrations of suspensionsamples were performed during the crystallization phase.

After the crystal suspension has been transferred to the filtrationunit, the liquid phase was pressed out through a filter cloth. Thefilter chamber was charged with a slowly increasing compression pressurethat was generated by a mechanical reduction of the volume of the filterchamber, and was slowly increased. The final filtration pressure reached10 bar. After filtration, the separated fractions were weighed. Theolein yield is the weight of the filtrate. The stearin yield is theweight of the crystal mass remaining on the filter. The yields of themeasured olein and stearin fractions are given in Table 7. Thecompositions of the feed materials, olein and stearin fractions aregiven in Table 8.

TABLE 7 Parameter Results Cooling curve (h) 13 Final Temperature of theSlurry (C.) 14.2 Solid Fat Content of the Slurry (%) 7.3 Solid FatContent of the Stearin (%) 39.6 Olein Yield (%) 83.4 Stearin Yield (%)14.4

TABLE 8 Parameter Feed Olein Stearin Moisture content (ppm) 564-660 — —Cloud point (° C.) 11.5-17.4 −4.8 to −5.5 — Iodine value 235.8-265   2604-278.7 184.2-210.8 Fatty acid composition (% w/w): 12:0 0.2-0.40.3-0.4 0.3-0.6 14:0 10.0-12.6 8.6-8.8 14.9-16.1 14:1 0.4-0.5 0.0-0.40.5-0.6 16:0 25.3-27.1 22.5-23.1 36.1-39.1 16:1 0.7-0.8 0.0 0.0 18:1n-90.3-1.9 0.3-0.5 0.0-0.4 22:1 0.9-1.0 1.0-1.1 0.7-0.8 20:5n-3 1.4-1.61.7-1.8 1.0-1.5 22:5n-6 14.6-17.1 18.0-18.3 11.9-12.9 22:6n-3 39.8-43.445.8-46.0 29.1-31.8 Solid fat content (%):  0° C. 8.7 0.0 36.3-44.1 10°C. 7.5 — 34.8-41.2 15° C. 6.8 — 33.2-38.5 20° C. 6.1 — 30.5-35.9 25° C.5.4 — 28.9-34.0 30° C. 3.1 — 26.3-31.1 35° C. 2.4 — 21.0-25.4 40° C. 0.8— 12.9-17.2 45° C. 0.0 — 4.5-5.2 50° C. 0.0 — 1.5-2.0 55° C. 0.0 — 0.0

The olein (liquid) and stearin (solid or semi-solid) fractions could befurther processed to produced deodorized oil by any of the minimalprocessing methods described herein and illustrated in the aboveexamples or by any method known in the prior art.

Example 5

The following Example shows a bench-scale process for forming a solidfat product of the invention.

An unwinterized oil extracted by hexane from biomass of a Schizochytriummicroorganism was heated to 40° C. until all solid material had meltedand a homogeneous liquid was formed. In a separate container,monoglyceride and diglyceride emulsifiers (DIMODAN PTK A, available fromDANISCO) were melted at the same temperature (40° C.) until completelyliquid. The melted emulsifiers were then added to the melted oil andmixed together. The oil/emulsifier mixture was then transferred to anice bath and mixed constantly while cooling. The cooled product wassolid with the consistency of shortening. The product was thentransferred to containers and stored.

Example 6

The following Example shows a bench-scale process for forming a solidfat product with the introduction of nitrogen during the step ofsolidifying the product.

The procedure was the same as discussed above in Example 5, except thatnitrogen was bubbled at a rate of between about 10 and about 50 ml/mininto the composition as it cooled, providing extra stability to theproduct and changing the color/physical characteristics of the productfrom a dull appearance to a shiny yellow appearance. The cooled productwas solid with the consistency of shortening. After cooling, the productwas transferred to containers and stored.

Example 7

The following Example shows a pilot or large-scale process for forming asolid fat product.

An unwinterized oil extracted by hexane from biomass of a Schizochytriummicroorganism was heated to 40° C. until all solid material melted and ahomogeneous liquid was formed. In a separate container, the emulsifiersas in Example 5 were melted at the same temperature (40° C.) untilcompletely liquid. The melted emulsifiers were then added to the meltedoil (HM) and mixed together. The hot liquid emulsion was introduced to abeaker in ice with stirring to emulate a scrape surface heat exchangerto cool. The composition was cooled from about 40° C. to about 0° C. inabout four minutes. During this process nitrogen was introduced into thecomposition at a rate of between about 10 and about 50 ml/min. Aftercooling, the resulting crystallized fat was then transferred tocontainers and stored.

Example 8

The following Example shows a pilot or large-scale process for forming asolid fat product with oil and nutrients added.

The mixture of emulsifier and oil was prepared as described in Example7. As the mixture cools, it was constantly mixed and water was added at5% by weight resulting in a product with a slightly differentconsistency. Ascorbic acid as a free acid was then added to the mixtureat about 5% by weight. Folic acid was then added to the mixture at about0.0008% by weight. The resulting crystallized fat then transferred tocontainers and stored.

Example 9

The following Example shows the increase in oxidative stability forcompositions of the present invention containing ascorbic acid andcontaining ascorbic acid and folic acid.

A solid fat composition of the invention was prepared in accordance withExample 5. Solid fat compositions additionally containing ascorbic acid(5 wt. %) and water (5 wt. %) and containing ascorbic acid (5 wt. %),folic acid (0.0008 wt. %) and water (5 wt. %) were prepared inaccordance with Example 1 by the introduction of the additionalingredients during the cooling step. The resulting compositions wereevaluated for oxidative stability and the results are shown in FIG. 3.As can be seen, the addition of ascorbic acid and water more thantripled the OSI value of the base composition. Further, the addition offolic acid to the composition with ascorbic acid and water increased theoxidative stability of the composition.

Example 10 Blended Compositions Containing Minimally Processed Oils

The DHA-rich oil from Example 3a and 3d above were blended with ARASCO®(Martek Biosciences, Columbia, Md.) in a ratio of 2:1 to produce ARA-and DHA-containing oil blends.

Quality Characteristics of Blended Composition Made from MinimallyProcessed DHA and ARASCO®

ARASCO ®/ ARASCO ® DHA oil (Ex. 2a), DHA oil (Ex. 2d), Parameter 2:1 2:1Physical Description: Color (Visual) Light Yellow Orange Color(Lovibond) 1.9 R/70.0 Y 3.2 R/70.0 Y Oil Clarity at 25° C. Viscousopaque liquid Viscous opaque liquid Oil Clarity at 40° C. Clear liquidClear liquid Chemical Analyses: DHA (mg/g) 123 127 DHA (area %) 12.913.3 ARA (mg/g) 247.3 255.6 ARA (area %) 27.3 28.1 PV (meq/kg) 0.45 0.45p-AV 5.9 7.2 FFA (%) 0.07 0.07 Moisture & Volatiles (%) Below detectionBelow detection Nonsaponifiables (%) 1.9 1.9 Trans Fatty Acids (%) Belowdetection Below detection Elemental Analyses: As Below detection Belowdetection Cu Below detection Below detection Fe Below detection Belowdetection Pb Below detection Below detection Hg Below detection Belowdetection Fatty Acid Profile (Major Fatty Acids): 14:0 5.2 4.0 16:0 17.416.7 18:0 5.7 5.8 18:1n-9 10.9 10.9 18:2n-6 4.6 4.8 18:3n-6 1.9 2.0 22:01.0 1.0 20:3n-6 2.3 2.3 20:4n-6 27.3 28.1 24:0 1.1 1.1 22:5n-6 4.8 5.022:6n-3 12.9 13.3

Example 11

The following example shows a bench-scale process for forming a solidfat product with oil from Schizochytrium and palm stearin.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was heated to 40-50° C. under nitrogenuntil all solid material melted and a homogeneous liquid was formed. Ina separate container, palm stearin (available from Ciranda Inc., Hudson,Wis.) was melted at the same temperature (40-50° C.) until completelyliquid. The ratio of unwinterized oil to palm stearin used was 75 to 25(%, w/w). Subsequently, melted palm stearin was mixed with unwinterizedoil from Schizochytrium. In another container, monoglyceride anddiglyceride emulsifiers (either Dimodan 930-KA or Grindsted PS 219/BK-A, available from Danisco, Denmark) were heated to 70-75° C. until ahomogeneous liquid was formed. The melted oil blend (unwinterized oiland palm stearin) was then added to the melted emulsifier and mixedtogether. The hot liquid formulation was cooled down to 15° C. in achiller batch under nitrogen and with agitation. Once thecrystallization temperature was reached, the formulation was held for 1hour at 15° C. with agitation. The resulting crystallized fatformulation was then transferred to containers and stored. The resultsare shown below in Table 9.

TABLE 9 Physical and Chemical Properties Results Peroxide value (meq/kg)4.1-8.6 Free fatty acids (%) 0.15-0.17 p-Anisidine value Below detectionRancimat (hr) 10.9-13.5 DHA content (mg/g) 215.4-239.2 Solid Fat Content(%): 10.0° C. 24.4-26.9 21.1° C. 18.3-19.8 26.7° C. 14.6-16.6 33.3° C. 9.2-10.6 37.8° C. 7.8-8.1

Example 12

The following example shows a bench-scale process for forming a solidfat product with oil from Schizochytrium and palm kernel stearin.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was heated to 40-50° C. under nitrogenuntil all solid material melted and a homogeneous liquid was formed. Ina separate container, palm kernel stearin (available from Ciranda Inc.,Hudson, Wis.) was melted at the same temperature (40-50° C.) untilcompletely liquid. The ratios of unwinterized oil to palm kernel stearinwere ranged from 75:25 to 80:20 (%, w/w). Subsequently, melted palmkernel stearin was mixed with unwinterized oil from Schizochytrium. Inanother container, monoglyceride and diglyceride emulsifiers (eitherDimodan 930-KA or Grindsted PS 219/B K-A, available from Danisco,Denmark) were heated to 70-75° C. until a homogeneous liquid was formed.The melted oil blend (unwinterized oil and palm kernel stearin) was thenadded to the melted emulsifier and mixed together. The hot liquidformulation was cooled down to 15° C. in a chiller batch under nitrogenand with agitation. Once the crystallization temperature has beenreached, the formulation was held for 1 hour at 15° C. with agitation.The resulting crystallized fat formulation was then transferred tocontainers and stored. The results are shown below in Table 10.

TABLE 10 Physical and Chemical Properties Results Peroxide value(meq/kg) 1.1 Free fatty acids (%) 0.11 p-Anisidine value Below detectionRancimat (hr) 7.3 DHA content (mg/g) 225.8 Solid Fat Content (%): 10.0°C. 32.6 21.1° C. 18.3 26.7° C. 11.2 33.3° C. 4.1 37.8° C. 2.0

Example 13

The following example shows a bench-scale process for forming a solidfat product containing Schizochytrium oil and palm kernel stearin withantioxidants added.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was mixed with 0.2% (w/w) antioxidants(containing 10% tocopherol and 10% ascorbyl palmitate) and heated to40-50° C. under nitrogen until all solid material melted and ahomogeneous liquid was formed. In a separate container, palm kernelstearin (available from Ciranda Inc., Hudson, Wis.) was melted at thesame temperature (40-50° C.) until completely liquid. The ratio ofunwinterized oil to palm kernel stearin was used at 75:25 (%, w/w).Subsequently, melted palm kernel stearin was mixed with unwinterized oilfrom Schizochytrium. In another container, monoglyceride and diglycerideemulsifiers (either Dimodan 930-KA or Grindsted PS 219/B K-A, availablefrom Danisco, Denmark) were heated to 70-75° C. until a homogeneousliquid was formed. The melted oil blend (unwinterized oil and palmkernel stearin) was then added to the melted emulsifier and mixedtogether. The hot liquid formulation was cooled down to 15° C. in achiller batch under nitrogen and with agitation. Once thecrystallization temperature has been reached, the formulation was heldfor 1 hour at 15° C. with agitation. The resulting crystallized fatformulation was then transferred to containers and stored. The resultsare shown below in Table 11.

TABLE 11 Physical and Chemical Properties Results Peroxide value(meq/kg) 0.3-0.5 Free fatty acids (%) 0.15-0.20 p-Anisidine value Belowdetection Rancimat (hr) 18.9-25.0 DHA content (mg/g) 232-243 Solid FatContent (%): 10.0° C. 30.6-35.6 21.1° C. 16.6-21.9 26.7° C.  9.7-15.233.3° C. 2.3-4.2 37.8° C. 1.5-3.2

Example 14

The following example shows a bench-scale process for forming a solidfat product with Schizochytrium oil and palm kernel stearin containing10-20% (w/w) DHA.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was mixed with 0.2% (w/w) antioxidants(containing 10% tocopherol and 10% ascorbyl palmitate) and heated to40-50° C. under nitrogen until all solid material melted and ahomogeneous liquid was formed. In a separate container, palm kernelstearin (available from Ciranda Inc., Hudson, Wis.) was melted at thesame temperature (40-50° C.) until completely liquid. The ratio ofunwinterized oil to palm kernel stearin was used at 60:40 (%, w/w).Subsequently, melted palm kernel stearin was mixed with unwinterized oilfrom Schizochytrium. In another container, monoglyceride and diglycerideemulsifiers (either Dimodan 930-KA or Grindsted PS 219/B K-A, availablefrom Danisco, Denmark) were heated to 70-75° C. until a homogeneousliquid was formed. The melted oil blend (unwinterized oil and palmkernel stearin) was then added to the melted emulsifier and mixedtogether. The hot liquid formulation was cooled down to 15° C. in achiller batch under nitrogen and with agitation. Once thecrystallization temperature has been reached, the formulation was heldfor 1 hour at 15° C. with agitation. The resulting crystallized fatformulation was then transferred to containers and stored. The resultsare shown below in Table 12.

TABLE 12 Physical and Chemical Properties Results Peroxide value(meq/kg) 0.5-0.7 Free fatty acids (%) 0.23-0.25 p-Anisidine value Belowdetection Rancimat (hr) 20-26 DHA content (mg/g) 192-199 Solid FatContent (%): 10.0° C. 39.8-42.6 21.1° C. 25.0-27.1 26.7° C. 13.6-12.233.3° C. 2.8-2.9 37.8° C. 2.5-3.0

Example 15

The following example shows a bench-scale process for forming a solidfat product with Schizochytrium oil and palm kernel stearin containing20-30% (w/w) DHA.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was mixed with 0.2% (w/w) antioxidants(consist of 10% tocopherol and 10% ascorbyl palmitate) and heated to40-50° C. under nitrogen until all solid material melted and ahomogeneous liquid was formed. In a separate container, palm kernelstearin (available from Ciranda Inc., Hudson, Wis.) was melted at thesame temperature (40-50° C.) until completely liquid. The ratios ofunwinterized oil to palm kernel stearin were ranged from 75:25 to 85:15(%, w/w). Subsequently, melted palm kernel stearin was mixed withunwinterized oil from Schizochytrium. In another container,monoglyceride and diglyceride emulsifiers (either Dimodan 930-KA orGrindsted PS 219/B K-A, available from Danisco, Denmark) were heated to70-75° C. until a homogeneous liquid was formed. The melted oil blend(unwinterized oil and palm kernel stearin) was then added to the meltedemulsifier and mixed together. The hot liquid formulation was cooleddown to 15° C. in a chiller batch under nitrogen and with agitation.Once the crystallization temperature has been reached, the formulationwas held for 1 hour at 15° C. with agitation. The resulting crystallizedfat formulation was then transferred to containers and stored. Theresults are shown below in Table 13.

TABLE 13 Physical and Chemical Properties Results Peroxide value(meq/kg) 0.0-0.6 Free fatty acids (%) 0.16-0.24 p-Anisidine value0.0-5.0 Rancimat (hr) 19.6-22.4 DHA content (mg/g) 236-283 Solid FatContent (%): 10.0° C. 27.7-32.1 21.1° C. 15.2-18.1 26.7° C.  9.8-11.633.3° C. 4.5-4.9 37.8° C. 2.7-2.9

Example 16

The following example shows a bench-scale process for forming a solidfat product containing Schizochytrium oil and palm kernel stearinwith >30% (w/w) DHA.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was mixed with 0.2% (w/w) antioxidants (10%tocopherol and 10% ascorbyl palmitate) and heated to 40-50° C. undernitrogen until all solid material melted and a homogeneous liquid wasformed. In a separate container, palm kernel stearin (available fromCiranda Inc., Hudson, Wis.) was melted at the same temperature (40-50°C.) until completely liquid. The ratio of unwinterized oil to palmkernel stearin used was 80:20 (%, w/w). Subsequently, melted palm kernelstearin was mixed with unwinterized oil from Schizochytrium. In anothercontainer, monoglyceride and diglyceride emulsifiers (either Dimodan930-KA or Grindsted PS 219/B K-A, available from Danisco, Denmark) wereheated to 70-75° C. until a homogeneous liquid was formed. The meltedoil blend (unwinterized oil and palm kernel stearin) was then added tothe melted emulsifier and mixed together. The hot liquid formulation wascooled down to 15° C. in a chiller batch under nitrogen and withagitation. Once the crystallization temperature has been reached, theformulation was held for 1 hour at 15° C. with agitation. The resultingcrystallized fat formulation was then transferred to containers andstored. The results are shown below in Table 14.

TABLE 14 Physical and Chemical Properties Results Peroxide value(meq/kg) 0.2-0.4 Free fatty acids (%) 0.12-0.17 p-Anisidine value Belowdetection Rancimat (hr) 18.9-19.9 DHA content (mg/g) 310-319 Solid FatContent (%): 10.0° C. 23.1-28.5 21.1° C. 11.4-16.5 26.7° C.  6.3-10.733.3° C. 4.3-5.0 37.8° C. 2.9-3.1

Example 17

The following example shows a bench-scale process for forming a solidfat product containing Schizochytrium oil and palm kernel stearin withNatural Butter Flavor and Bitterness Masker added.

An unwinterized fully refined oil produced from biomass of aSchizochytrium microorganism was mixed with 0.2% (w/w) antioxidants (10%tocopherol and 10% ascorbyl palmitate), 0.1-0.15% (w/w) natural butterflavor (available from Danisco) and 0.03-0.05% (w/w) natural bitternessmasker (available from Firmenich Inc.). It was then heated to 40-50° C.under nitrogen until all solid material melted and a homogeneous liquidwas formed. In a separate container, palm kernel stearin (available fromCiranda Inc., Hudson, Wis.) was melted at the same temperature (40-50°C.) until completely liquid. The ratio of unwinterized oil to palmkernel stearin used was 80:20 (%, w/w). Subsequently, melted palm kernelstearin was mixed with unwinterized oil from Schizochytrium. In anothercontainer, monoglyceride and diglyceride emulsifiers (Dimodan 930-KA,available from Danisco, Denmark) were heated to 70-75° C. until ahomogeneous liquid was formed. The melted oil blend (unwinterized oiland palm kernel stearin) was then added to the melted emulsifier andmixed together. The hot liquid formulation was cooled down to 15° C. ina chiller batch under nitrogen and with agitation. Once thecrystallization temperature has been reached, the formulation was heldfor 1 hour at 15° C. with agitation. The resulting crystallized fatformulation was then transferred to containers and stored. The resultsare shown below in Table 15.

TABLE 15 Physical and Chemical Properties Results Peroxide value(meq/kg) Below detection Free fatty acids (%) 0.19-0.2  p-Anisidinevalue Below detection Rancimat (hr) 28.3-28.5 DHA content (mg/g) 230-283Solid Fat Content (%): 10.0° C. 21.8-27.9 21.1° C. 14.0-14.8 26.7° C.7.2-8.6 33.3° C. 2.6-4.0 37.8° C. 1.9-2.2

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein should not,however, be construed as limited to the particular forms disclosed, asthese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the present invention. Accordingly, theforegoing best mode of carrying out the invention should be consideredexemplary in nature and not as limiting to the scope and spirit of theinvention as set forth in the appended claims.

1-98. (canceled)
 99. A method of preparing an oil product that is usedfor consumption, comprising: a) extracting an oil-containing fractionfrom a microbial biomass, wherein the oil-containing fraction comprisesat least one LC-PUFA and saturated fatty acids at least sufficient tovisually affect the oil-containing fraction; and b) treating theoil-containing fraction by vacuum evaporation to produce an oil productcomprising at least one LC-PUFA; wherein the oil product has not beensubject to a winterization step.
 100. (canceled)
 101. (canceled) 102.(canceled)
 103. The method of claim 99, wherein the oil product has notbeen subject to a caustic refining process, a chill filtration process,or a bleaching process.
 104. The method of claim 99, wherein themicrobial biomass is from a microorganism selected from the groupconsisting of microorganisms of the genus Thraustochytrium,microorganisms of the genus Schizochytrium, microorganisms of the genusAlthornia, microorganisms of the genus Aplanochytrium, microorganisms ofthe genus Japonochytrium, microorganisms of the genus Elina,microorganisms of the genus Crypthecodinium, and mixtures thereof. 105.The method of claim 104, wherein the microorganism is selected from thegroup consisting of microorganisms of the genus Schizochytrium,microorganisms of the genus Crypthecodinium, and mixtures thereof. 106.The method of claim 99, wherein the oil-containing fraction comprises anLC-PUFA having a carbon chain length of at least
 20. 107. (canceled)108. The method of claim 106, wherein the LC-PUFA has at least threedouble bonds.
 109. (canceled)
 110. The method of claim 106, wherein theLC-PUFA comprises an LC-PUFA selected from the group consisting ofdocosahexaenoic acid, docosapentaenoic acid, arachidonic acid, andeicosapentaenoic acid.
 111. (canceled)
 112. (canceled)
 113. (canceled)114. The method of claim 99, wherein the step of treating theoil-containing fraction comprises desolventization.
 115. The method ofclaim 114, wherein the desolventization comprises subjecting theextracted oil-containing fraction to vacuum conditions at hightemperature.
 116. (canceled)
 117. The method of claim 115, wherein thedesolventization comprises subjecting the extracted oil-containingfraction to a vacuum of greater than a vacuum of about 100 mm Hg. 118.(canceled)
 119. (canceled)
 120. The method of claim 99, wherein the stepof treating the oil-containing fraction comprises deodorization. 121.The method of claim 120, wherein the deodorization comprises subjectingthe extracted oil-containing fraction to vacuum conditions at hightemperature while sparging the extracted oil-containing fraction withsteam.
 122. (canceled)
 123. (canceled)
 124. (canceled)
 125. (canceled)126. The method of claim 99, wherein the oil product has a free fattyacid content of less than about 0.5 wt. %.
 127. (canceled)
 128. Themethod of claim 99, wherein the oil product has a phosphorous value ofless than about 10 ppm.
 129. (canceled)
 130. The method of claim 99,wherein the oil product has a peroxide value of less than about 2meq/kg.
 131. (canceled)
 132. The method of claim 99, wherein the oilproduct has an anisidine value of less than about
 5. 133. (canceled)134. The method of claim 99, wherein the oil product has a soap contentof less than about 5 wt. %.
 135. (canceled)
 136. The method of claim 99,wherein the oil product has an Fe concentration of less than about 1ppm.
 137. (canceled)
 138. The method of claim 99, wherein the oilproduct has a Pb concentration of less than about 1 ppm.
 139. (canceled)140. The method of claim 99, wherein the oil product has an Hgconcentration of less than about 0.1 ppm.
 141. (canceled)
 142. Themethod of claim 99, wherein the oil product has an Ni concentration ofless than about 0.1 ppm.
 143. (canceled)
 144. The method of claim 99,wherein the oil product has a Cu concentration of less than about 1 ppm.145. (canceled)
 146. The method of claim 99, wherein the oil product hasbeen subjected to the step of bleaching either before or after the stepof treating.
 147. The method of claim 99, further comprisingfractionating the oil into an olein fraction and a stearin fraction.148. The method of claim 99, wherein the oil product is used for humanconsumption.
 149. The method of claim 99, wherein the oil product is asolid at 20° C.
 150. An oil product produced by the process of claim 99.151. A microbial oil product that is used for consumption prepared byextracting an oil-containing fraction from a microbial biomass, whereinthe oil-containing fraction comprises at least one LC-PUFA and saturatedfatty acids at least sufficient to visually affect the oil-containingfraction, and treating the fraction by a process of vacuum evaporation,wherein the oil product is not subjected to a winterization step. 152.(canceled)
 153. (canceled)
 154. (canceled)
 155. (canceled) 156.(canceled)
 157. (canceled)
 158. (canceled)
 159. (canceled) 160.(canceled)
 161. (canceled)
 162. (canceled)
 163. (canceled) 164.(canceled)
 165. (canceled)
 166. (canceled)
 167. (canceled) 168.(canceled)
 169. (canceled)
 170. (canceled)
 171. (canceled) 172.(canceled)
 173. (canceled)
 174. (canceled)
 175. (canceled) 176.(canceled)
 177. (canceled)
 178. (canceled)
 179. (canceled) 180.(canceled)
 181. (canceled)
 182. (canceled)
 183. A microbial oil productthat is used for consumption prepared by a process, comprising: a)extracting an oil-containing fraction comprising at least one LC-PUFAfrom a microbial biomass; and b) treating the fraction by a process ofvacuum evaporation; wherein the oil product is not subjected to awinterization step, a caustic refining process, a chill filtrationprocess, or a bleaching process; and wherein the oil product has acharacteristic selected from the group consisting of a free fatty acidcontent of less than about 0.5 wt. %, a phosphorous value of less thanabout 10 ppm, a peroxide value of less than about 2 meq/kg, an anisidinevalue of less than about 5, a soap content of less than about 5 wt. %,an Fe concentration of less than about 1 ppm, a Pb concentration of lessthan about 1 ppm, an Hg concentration of less than about 0.1 ppm, an Niconcentration of less than about 0.1 ppm, and a Cu concentration of lessthan about 1 ppm.
 184. (canceled)
 185. (canceled)
 186. (canceled) 187.(canceled)
 188. (canceled)
 189. A method of preparing an oil productthat is used for consumption, comprising: a) extracting anoil-containing fraction from a microbial biomass, wherein theoil-containing fraction comprises at least one LC-PUFA; and b) treatingthe oil-containing fraction by vacuum evaporation to produce an oilproduct comprising at least one LC-PUFA; wherein the oil product has notbeen subject to a caustic refining process.
 190. (canceled) 191.(canceled)
 192. (canceled)
 193. (canceled)
 194. A blended oil product,comprising the oil product of claim 151 and the oil product of claim193.
 195. (canceled)
 196. (canceled)
 197. (canceled)
 198. (canceled)199. A process to produce a liquid LC PUFA-containing oil fraction andan LC PUFA-containing solid fat product, comprising: fractionating amicrobial crude oil into an oil product and a solid fat product, whereinthe solid fat product has an LC PUFA content of at least about 20% byweight.
 200. (canceled)
 201. (canceled)